Urinary excretion of three nucleic acid oxidation adducts and isoprostane F(2)alpha measured by liquid chromatography-mass spectrometry in smokers, ex-smokers, and nonsmokers

To assess novel liquid chromatography/mass spectrometric methods for measuring oxidative damage to nucleic acids and lipids, we compared urinary excretion of 8-hydroxy-2'-deoxyguanosine (8-OHdG), 5-hydroxymethyl-2'-deoxyuridine (5-OHmU), and 8-hydroxyguanosine (8-OxoG), and an isoprostane, 8-iso-prostaglandin F(2)alpha (IsopF(2)alpha) in 234 healthy men (n = 113) and women (n = 121), 80 current smokers, 96 never-smokers), and 58 ex-smokers (no tobacco use for 3 years). The 8-OHdG and 8-OxoG did not differ significantly by group; 5-OHmU was higher in smokers, compared with ex- (p <.003) and never- (p <.0001) smokers and in ex- vs. never-smokers (p =.014) at, respectively, 13.5 +/- 0.7, 11.3 +/- 1.0, and 8.7 +/- 0.3 microg/g creatinine. IsopF(2)alpha was higher in smokers, compared with ex- (p =.007) and never-smokers (p <.0001) and in ex- vs. never- smokers (p =.002) at, respectively, 1.1 +/- 0.10; 0.74 +/- 0.07, and 0.51 +/- 0.04 microg/g creatinine. There were significant correlations among all three nucleic acid adducts and between IsopF(2)alpha and both 5-OHmU and 8-OHdG. Many smokers and ex-smokers had high levels of either 5-OHmU excretion or IsopF(2)alpha excretion, but not both. We conclude that 5-OHmU and IsopF(2)alpha are more discriminating of oxidative stress from tobacco smoke than the other two compounds measured. Whether characteristic patterns of excretion of these indicators forecast differential disease risk should be explored in future research.     

Oxidative stress in very low birth weight infants as measured by urinary 8-OHdG

Very low birth weight (VLBW) infants can be subjected to oxidative stress in the course of intensive care. We measured 8-hydroxydeoxyguanosine (8-OHdG), a biomarker of oxidative stress, and estimated the degree of oxidative stress in such infants. We also examined if the administered oxygen was related to oxidative stress. Urine samples of 50 Japanese VLBW infants [birth weights: 956.3+/-277.6g, and gestational ages: 28.0+/-2.6 weeks (mean +/- SD)] were collected on various postnatal days and 8-OHdG levels were determined using an ELISA kit. Sixteen term infants served as normal controls. As body weights at sampling increased, the average levels of urinary 8-OHdG decreased. 8-Hydroxydeoxyguanosine levels were: infants under 1000g, 29.5+/-16.4 micromol/mol creatinine (n = 24); 1000-1500g, 23.8+/-14.9 (n = 12); over 1500g, 16.1+/-8.5 (n = 14); and control, 10.9+/-7.2 (n = 16). Significant differences were found between <1000g group and > or = 1500g group (p = 0.0030), <1000g group and control (p < 0.0001), and 1000-1500g group and control (p = 0.0108). Also as postconceptional age at sampling increased, the average levels of 8-OHdG decreased. 8-Hydroxydeoxyguanosine levels were: infants before 252 days (36 weeks) of postconception: 27.4+/-15.5 micromol/mol creatinine (n = 34); after 252 days, 18.2+/-12.5 (n = 16). Differences between <252 days group and control (p < 0.0001), and <252 days group and > or = 252 days groups (p = 0.0253) were statistically significant. Among the three groups based on ambient oxygen concentration (21%, 22-29%, and > or = 30%) there was no significant difference (p = 0.417). The more premature the infants were, the more intense was the oxidative stress, hence, it is the prematurity rather than the administered oxygen which causes oxidative stress in VLBW infants. Drury et al. ["Urinary 8-hydroxydeoxyguanosine in infants and children" Free Radic. Res. 28 (1998) 423-4281 measured urinary 8-OHdG of 28 infants (24-40 weeks gestation) and found no gestation or birthweight related differences. This discrepancy seemed to be because of difference in birth weights and sampling period of the subjects.     

Urinary excretion of 8-hydroxy-2'-deoxyguanosine measured by high-performance liquid chromatography with electrochemical detection

There is good evidence that oxidative DNA damage permanently occurs in living cells. The oxidative DNA damage product 8-hydroxy-2'-deoxyguanosine (8-OHdG) is one of the predominant forms of radical-induced lesions to DNA, and has therefore been widely used as a biomarker for oxidative stress, either in cellular DNA or as DNA repair product in urine. In this paper we describe the use of a high-performance liquid chromatographic procedure with electrochemical detection for the measurement of urinary 8-OHdG. Our study has addressed the questions (i) of baseline urinary levels of 8-OHdG in spot urine and 24-h urine, (ii) of inter- and intra-individual variation of this biomarker, and (iii) of confounding factors for the excretion of 8-OHdG. No significant difference between the mean group levels of 8-OHdG/creatinine in spot urine (2.03+/-1.21 micromol/mol, n=148) and in 24-h urine (1.86+/-1.09 micromol/mol, n=67) was observed. However, when only 24-h urine was used for analysis, 8-OHdG was found to be statistically significantly higher in smokers. By multiple linear regression analysis, urinary creatinine was identified as the only predictor of 8-OHdG/24 h (r(p)=0.33, P=0.007). High intra-individual coefficients of variation of 8-OHdG/24 h were observed in two healthy subjects over a period of 10 consecutive days (37 and 57%, respectively), indicating that the intra-individual fluctuation of urinary 8-OHdG has so far been underestimated. Therefore, we suggest that single values of 8-OHdG should be considered with caution, in particular in small study groups and when spot urine is used.     

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.     

Plasma 3-nitrotyrosine,urinary 8-isoprostane and 8-OHdG among healthy Japanese people

To examine the relation between lifestyle factors and oxidative stress biomarkers, this study analysed plasma 3-nitrotyrosine (3-NT), urinary 8-isoprostane and 8-hydroxy-2’- deoxyguanosine (8-OHdG) of 323 healthy Japanese without any disease. Plasma 3-NT was significantly increased by excessive exercise (p=0.010), but it was not significantly different in terms of sex, age (< 40, ≧40), BMI (<18.5, 18.5–24.9, ≧25.0), smoking (non-smokers, smokers) and alcohol drinking per week (non-drinkers, <10 units, ≧10 units). Urinary 8-isoprostane was significantly associated with alcohol drinking (p <0.01) and sex (p <0.01), although it had no significant relevance to age and exercise. Moreover, urinary 8-OHdG was positively associated with age (p <0.05) and negatively associated with BMI (p <0.05) and fasting insulin (p <0.001). However, it was not related with sex, smoking, alcohol drinking and exercise. In conclusion, the present results suggest that 3-NT, 8-isoprostane and 8-OHdG seem to be useful biomarkers for early prediction of lifestyle-related disease risk at the population level.     

Cigarette smokers have decreased lymphocyte and platelet alpha-tocopherol levels and increased excretion of the gamma-tocopherol metabolite gamma-carboxyethyl-hydroxychroman (gamma-CEHC)

Cigarette smoking is associated with increased oxidative stress and increased risk of degenerative disease. As the major lipophilic antioxidant, requirements for vitamin E may be higher in smokers due to increased utilisation. In this observational study we have compared vitamin E status in smokers and non-smokers using a holistic approach by measuring plasma, erythrocyte, lymphocyte and platelet alpha- and gamma-tocopherol, as well as the specific urinary vitamin E metabolites alpha- and gamma-carboxyethyl-hydroxychroman (CEHC). Fifteen smokers (average age 27 years, smoking time 7.5 years) and non-smokers of comparable age, gender and body mass index (BMI) were recruited. Subjects completed a 7-day food diary and on the final day they provided a 24 h urine collection and a 20 ml blood sample for measurement of urinary vitamin E metabolites and total vitamin E in blood components, respectively. No significant differences were found between plasma and erythrocyte alpha- and gamma-tocopherol in smokers and non-smokers. However, smokers had significantly lower alpha-tocopherol (mean+/-SD, 1.34+/-0.31 micromol/g protein compared with 1.94+/-0.54, P = 0.001) and gamma-tocopherol (0.19+/-0.04 micromol/g protein compared with 0.26+/-0.08, P = 0.026) levels in their lymphocytes, as well as significantly lower alpha-tocopherol levels in platelets (1.09+/-0.49 micromol/g protein compared with 1.60+/-0.55, P = 0.014; gamma-tocopherol levels were similar). Interestingly smokers also had significantly higher excretion of the urinary gamma-tocopherol metabolite, gamma-CEHC (0.49+/-0.25mg/g creatinine compared with 0.32+/-0.16, P = 0.036) compared to non-smokers, while their alpha-CEHC (metabolite of alpha-tocopherol) levels were similar. There was no significant difference between plasma ascorbate, urate and F2-isoprostane levels. Therefore in this population of cigarette smokers (mean age 27 years, mean smoking duration 7.5 years), alterations to vitamin E status can be observed even without the more characteristic changes to ascorbate and F2-isoprostanes. We suggest that the measurement of lymphocyte and platelet vitamin E may represent a valuable biomarker of vitamin E status in relation to oxidative stress conditions.     

Increased local matrix metalloproteinase-8 expression in the periodontal connective tissues of smokers with periodontal disease

Matrix metalloproteinase (MMP)-8 has been associated with the progression of periodontitis, a common inflammatory disease of the supporting structures of the teeth, and with other degradative diseases. Tobacco smokers are at high risk of developing periodontitis that may progress more rapidly and respond poorly to treatment. Therefore, MMP-8 expression was determined by immunofluorescence staining in 60 random, computer-selected fields in the excised periodontal tissues of smokers and non-smokers, balanced for age, gender, and periodontal status. Immunofluorescence intensity, representing MMP-8 expression, in the periodontal tissues of smokers (30 fields from 6 subjects, mean 1154+/-124 units) was significantly higher than that in the periodontal tissues of non-smokers (30 fields from 6 subjects, mean 817+/-60 units; p < 0.05). Serum MMP-8 concentrations were measured by ELISA and compared in a larger group of smokers (n = 20) and age- and gender-balanced non-smokers (n = 20). Systemic MMP-8 concentrations in smokers and non-smokers were not significantly different (p > 0.05). A local tobacco-related increase in MMP-8 burden may contribute to periodontal disease progression in tobacco smokers. This finding may also have relevance to other tobacco-induced inflammatory diseases, such as vascular and pulmonary diseases.     

Association of serum arginase I with oxidative stress in a healthy population

The association of serum arginase I with oxidative stress was evaluated cross-sectionally in a healthy population. The mean levels of serum arginase I in healthy people (n = 278) were 32.6 ± 22.3 ng/ml. Significant correlations of arginase I were observed with age, WBC, RBC, alanine aminotransferase (ALT), high-sensitivity C-reactive protein (hs-CRP), uric acid, body mass index (BMI) and urinary 8-isoprostane. Multiple regression analysis showed significant associations of arginase I with WBC, RBC, urinary 8-hydroxydeoxyguanosine (8-OHdG), age, HbA1c and urinary 8-isoprostane. In the associations of arginase I with 8-OHdG, 8-isoprostane and HbA1c, confounding factors and lifestyle factors such as sex, old age, smoking and alcohol consumption were involved. It was concluded that serum arginase I was associated with oxidative stress and HbA1c in addition to age, WBC and RBC in healthy Japanese people and may become a new biomarker for early prediction of diabetes mellitus and other oxidative stress-related diseases.     

Assessment of DNA damage and its modulation by dietary and genetic factors in smokers using the Comet assay: a biomarker model.

Methods are needed to assess exposure to genotoxins in humans and to improve understanding of dietary cancer prevention. The Comet assay was used to detect smoking-related exposures and dietary modulations in target tissues. Buccal scrapings, blood and faeces were collected from 38 healthy male volunteers (smokers and non-smokers) during a dietary intervention study with bread supplemented with prebiotics+/-antioxidants. GSTM1-genotype was determined with PCR. Buccal and peripheral lymphocytes were analysed for DNA damage using the Comet assay. Genotoxicity of faecal water (FW) was assayed in human colon HT29 clone 19A cells. 'Tail intensity' (TI) was used as a quantitative indicator of DNA damage in the Comet assay. Intervention with bread reduced DNA damage in lymphocytes of smokers (8.3+/-1.7% TI versus 10.2+/-4.1% TI, n=19), but not of non-smokers (8.6+/-2.8% TI versus 8.3+/-2.7% TI, n=15). Faecal water genotoxicity was reduced only in non-smokers (9.4+/-2.9% TI versus 18.9+/-13.1% TI, n=15) but not in smokers (15.5+/-10.7% TI versus 20.4+/-14.1% TI, n=13). The Comet assay was efficient in the detection of both smoking-related exposure (buccal cells) and efficacy of dietary intervention (faecal samples). Smokers and non-smokers profited differently from the intervention with prebiotic bread+/-antioxidants. Stratification of data by genotype enhanced specificity/sensitivity of the intervention effects and contributed important information on the role of susceptibility.     

Genotype dependent and cigarette specific effects on endothelial nitric oxide synthase gene expression and enzyme activity

We explored the interactive effects of endothelial nitric oxide synthase (eNOS) genotypes and cigarette smoking on protein levels and enzyme activity in 33 postpartum placentas. Whilst the eNOS protein levels were lower in the rare allele (0.48+/-0.11, n=9 vs. 1. 05+/-0.10, n=24, P<0.01), the eNOS enzyme activity was about 7-fold higher in the rare allele (4556.2+/-255.4 vs. 621.8+/-180.5 cpm/mg/min, P<0.01). Smokers had lower eNOS protein levels (1.07+/-0. 09 vs. 0.50+/-0.19, P<0.05) in both alleles. It reduced the eNOS activities only in the rare allele (non-smokers: 6143.8+/-251.2, n=5, smokers: 2968.5+/-259.4, n=4, 52% reduction, P<0.01). We conclude that associations between eNOS polymorphism and protein levels and enzyme activities are modifiable by smoking, the effects of smoking are dependent on the eNOS genotypes.     

Comprehensive characterisation of pulmonary and serum surfactant protein D in COPD

Background

Pulmonary surfactant protein D (SP-D) is considered as a candidate biomarker for the functional integrity of the lung and for disease progression, which can be detected in serum. The origin of SP-D in serum and how serum concentrations are related to pulmonary concentrations under inflammatory conditions is still unclear.

Methods

In a cross-sectional study comprising non-smokers (n = 10), young - (n = 10), elderly smokers (n = 20), and smokers with COPD (n = 20) we simultaneously analysed pulmonary and serum SP-D levels with regard to pulmonary function, exercise, repeatability and its quaternary structure by native gel electrophoresis. Statistical comparisons were conducted by ANOVA and post-hoc testing for multiple comparisons; repeatability was assessed by Bland-Altman analysis.

Results

In COPD, median (IQR) pulmonary SP-D levels were lower (129(68) ng/ml) compared to smokers (young: 299(190), elderly: 296(158) ng/ml; p < 0.01) and non-smokers (967(708) ng/ml; p < 0.001). The opposite was observed in serum, with higher concentrations in COPD (140(89) ng/ml) as compared to non-smokers (76(47) ng/ml; p < 0.01). SP-D levels were reproducible and correlated with the degree of airway obstruction in all smokers. In addition, smoking lead to disruption of the quaternary structure.

Conclusions

Pulmonary and serum SP-D levels are stable markers influenced by smoking and related to airflow obstruction and disease state. Smaller subunits of pulmonary SP-D and the rapid increase of serum SP-D levels in COPD due to exercise support the translocation hypothesis and its use as a COPD biomarker.

Trial registration

no interventional trial     

The influence of smoking on plasma folate and lipoproteins in Alzheimer disease, mild cognitive impairment and depression

The risk for Alzheimer's disease (AD) is associated with lifestyle factors, especially cigarette smoking. In this study we investigated the influence of smoking on the serum levels of folic acid, LDL and HDL in AD patients, patients with minimal cognitive impairment (MCI) and patients with major depression. We investigated a total of n = 374 patients in the diagnostic categories:, AD: n = 272, MCI: n = 60, Major depression: n = 42. We found significantly lower HDL levels in smokers and previous smokers in comparison to non-smokers, p<0,05. The LDL: HDL ratio in smokers was significant higher (+20%) compared to previous smokers and non-smokers, p < 0.05. The mean levels of folic acid were statistically significant (p<0.05) lower (-24%) in smokers compared to non-smokers. Patients with MCI and Alzheimer;s disease (and also major depression) who are "smokers" show serum levels of HDL and folic acid that are known to be strong risk factors for vascular damage and increased risk for vascular brain damage and impaired cognitive function. Therefore cessation of smoking, substitution with folate or statin therapy of smoking patients with MCI or AD might be beneficial to slow down further cognitive decline.     

Impact of smoking on the metabolic action of subcutaneous regular insulin in type 2 diabetic patients.

BACKGROUND AND AIMS: Smoking has repeatedly been associated with alterations in both insulin sensitivity and insulin absorption in type 2 diabetes, which should lead to differences in the pharmacokinetic (PK) and pharmacodynamic (PD) properties of regular insulin (RI). However, a direct comparison of the PK/PD-effects of RI has never been performed in these patients. Therefore, the aim of this exploratory study was to investigate the time-action profile of RI in a small group of smoking and matching non-smoking patients with type 2 diabetes using the euglycemic glucose clamp technique, which is seen as the gold standard for PD/PK investigations. MATERIAL AND METHODS: Nine smokers (more than 10 cigarettes per day) and nine non-smokers matched for gender, age, and BMI (without significant differences in HbA (1c), diabetes duration or blood pressure) were enrolled in the study. Patients' blood glucose was stabilized overnight at 7.2 mmol/l using a Biostator. Smokers were required to smoke one cigarette within ninety minutes prior to injection of 18 U RI s. c. in the morning. Glucose infusion rates (GIR) were registered for the subsequent 480 min. RESULTS: Injection of 18 U of RI resulted in significantly higher insulin concentrations in smokers compared to non-smokers, in particular in the later part of the experiment (Insulin-AUC (240-480) 10.5 +/- 2.3 (mean +/- SD) vs. 7.8 +/- 1.6 microU/ml/min, p < 0.05). This was also reflected in the PD results with a higher metabolic effect in smokers in the last four hours of the experiment (GIR-AUC (240-480) 0.9 +/- 0.4 vs. 0.6 +/- 0.3 g/kg, p < 0.05). Pharmacokinetic analyses revealed a trend towards a lower insulin clearance in smokers (1.1 +/- 0.2 vs. 1.4 +/- 0.4 l/min, p = 0.08). CONCLUSIONS: This pilot study conducted in a small group of patients with type 2 diabetes shows that regular insulin exhibits a longer-lasting rise in insulin concentrations and a higher metabolic effect four to eight hours after injection in smokers compared to non-smokers. This suggests that hyperinsulinemia in smoking type 2 diabetic patients is at least partly caused by a deterioration in insulin clearance.     

Evaluation of a multi-parameter biomarker set for oxidative damage in man: increased urinary excretion of lipid, protein and DNA oxidation products after one hour of exercise

The objective of the present study was to evaluate a comprehensive set of urinary biomarkers for oxidative damage to lipids, proteins and DNA, in man. Eighteen moderately trained males (mean age 24.6+/-0.7) exercised 60min at 70% of maximal O2 uptake on a cycle ergometer. Urine fractions for 12 h were collected 1 day before, and for 3 consecutive days after exercise. As biomarkers of lipid peroxidation, 8 aldehydes (i.e. propanal, butanal, pentanal, hexanal, heptanal, octanal, nonanal and malondialdehyde-MDA)and acetone were analyzed in urines by gas chromatography with electron capture detection (GC-ECD). As a biomarker of protein oxidation, o,o'-dityrosine was analyzed in urine samples by a recently developed isotope dilution HPLC-atmospheric pressure chemical ionization (APCI)-tandem-mass spectrometry (HPLC-APCI-MS/MS) methodology. As a biomarker of oxidative DNA damage, urinary excretion of 8-hydroxy-2'-deoxyguanosine (8-OHdG) was measured by an ELISA method. On the day of exercise, significant increases were observed in urinary excretions of acetone (p < 0.025, n = 18) and butanal (p < 0.01, n = 18) in the 12h daytime fractions compared to the daytime fraction before exercise. The urinary acetone excretion was also significantly (p < 0.05) increased on the 1st day after exercise. Octanal and nonanal were increased in the daytime urine fraction on the 2nd day after exercise. However, these increases were of borderline significance (p = 0.09 and p = 0.07, respectively). Significantly elevated urinary o,o'-dityrosine amounts were observed in the daytime fraction on the day of exercise (p < 0.025) and on the 1st day after exercise (p = 0.07) compared to the before exercise daytime fraction. Excretion of urinary 8-OHdG was statistically significantly increased in the daytime fractions on the day of exercise (p = 0.07) and on the 1st day after exercise (p < 0.025) compared to before exercise daytime fraction. Increases in urinary excretions of acetone, propanal, pentanal, MDA and 8-OHdG significantly correlated with training status (hours of exercise/week) of the volunteers, while o,o'-dityrosine did not. To our knowledge, the present study is the first to evaluate a multi-parameter non-invasive biomarker set for damage to three main cellular targets of ROS. It shows that 1 h of exercise may already induce oxidative damage in moderately trained individuals and that the chosen urinary biomarkers are sensitive enough to monitor such damage.     

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.     

Urinary excretion of 8-hydroxydeoxyguanosine and malondialdehyde after high dose radiochemotherapy preceding stem cell transplantation

The urinary excretion of the hydroxylated DNA base 8-hydroxydeoxyguanosine (8-OHdG) and the lipid peroxidation product malondialdehyde (MDA) was monitored in 11 patients with hematological malignancies undergoing total body irradiation and high-dose chemotherapy preceding bone marrow transplantation. Nine patients showed a prompt increase in urinary 8-OHdG (8-25 times the initial baseline level) on days 0-7 after irradiation onset; the excretion then decreased during the aplastic period and increased again when engraftment took place (in 7 patients). A significant positive correlation was found between urinary 8-OHdG and whole blood leukocyte count, both on day 5 (p =.04, r =.72) and on day 22 (p =.009, r =.80) after irradiation onset. One patient who lacked the first peak of 8-OHdG excretion showed low blood leukocyte counts (less than 2 x 10(9)/l) before therapy onset; this patient, however, later had a successful engraftment and then also showed considerable increases in both 8-OHdG excretion and leukocyte count. These observations suggest leukocytes play a part in the excretion of 8-OHdG after conditioning therapy preceding bone marrow transplantation. As opposed to the biphasic 8-OHdG excretion, the excretion of MDA showed a single peak appearing on days 11-19 after radiochemotherapy onset, i.e., during the period in which the patients suffered from cytopenia, mucositis, and other side effects of the treatment. It is suggested, therefore, that these clinical manifestations are associated with increased lipid peroxidation. Altogether, these findings illustrate the utility of serial urinary samples for monitoring oxidative stress due to conditioning therapy in clinical practice. They also demonstrate that different oxidative stress markers may behave quite differently regarding their appearance in the urine after whole-body oxidative stress.     

Are plasma insulin-like growth factor I (IGF-I) and IGF-binding protein 3 (IGFBP-3) useful markers of prostate cancer?

Increased concentrations of insulin-like growth factor I (IGF-I) and decreased insulin-like growth factor binding protein 3 (IGFBP-3) in serum have been proposed as markers of prostate cancer (CaP). The evidence for this, however, is contradictory. We assayed serum for IGF-I, IGFBP-3 and prostate-specific antigen (PSA) in patients with CaP and benign prostatic hyperplasia (BPH) and in healthy controls (HC). The mean +/- SD concentration of IGF-I in CaP (98.3 +/- 39.3 ng/mL; n = 15) was lower than in BPH (119 +/- 31.1 ng/mL; n=24) and HC (119 +/- 36.1 ng/mL; n=46), but the differences between the three groups were not statistically significant (p > 0.05). The mean IGFBP-3 concentrations in CaP (2691 +/- 1105 ng/mL; n = 16; p = 0.029) and BPH (2618 +/- 816 ng/mL; n = 26; p = 0.006) patients were significantly lower than that of the HC (3119 +/- 618 ng/mL; n=59), but the difference between the two groups of patients was not significant (p > 0.05). PSA concentrations in CaP (median = 80.8 ng/mL; n = 25) were significantly higher than those in BPH (median = 8.6 ng/mL; n = 39) (p < 0.001). Ninety-six percent of CaP and 72% of BPH patients had PSA concentrations >4.0 ng/mL; the proportions of patients with concentrations exceeding 20 ng/mL were 76% and 10%, respectively. We conclude that IGF-I and IGFBP-3 are inferior to PSA for CaP detection.     

Altered salivary profile in heavy smokers and its possible connection to oral cancer

Saliva is the first biological fluid to encounter inhaled cigarette smoke, whose numerous carcinogens and oxidants are responsible for the oral cancer so prevalent among smokers. Whole saliva, collected from 25 consenting heavy smokers and from a control group of 25 age- and gender-matched non-smokers, was subjected to sialochemical, biochemical, immunological and oxidative analyses. The mean flow rate was significantly higher in smokers than in non-smokers, as were the median activity value of superoxide dismutase (SOD) and the total salivary antioxidant capacity (ImAnOx) (by 32% and 12%, respectively, p=0.05). The salivary carbonyl concentration (an oxidative stress indicator) was significantly higher by 126% (p=0.0006) among smokers, while lactate dehydrogenase, albumin, total immunoglobulin G, and the metalloproteinases MMP-2 and MMP-9 concentrations were significantly lower in the smokers, by 86% (p=0.003), 65% (p=0.003), 61% (p=0.048), 35% (p=0.005) and 55% (p=0.035), respectively. Apparently, the oral cavity's salivary antioxidant system fails to cope with the severe attack of reactive oxygen species originating in cigarette smoke. Moreover, various other salivary functional and protective parameters also decreased among the smokers. Hence, further research aimed at examining the possibility of administration of agents as antioxidants or saliva substitutes to the oral cavity of smokers should be considered.     

Biomonitoring of environmental tobacco smoke (ETS)-related exposure to 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)

The exposure of non-smokers to the tobacco-specific N-nitrosamine 4-(N-methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), a rodent lung carcinogen, was determined in the air of various indoor environments as well as by biomonitoring of non-smokers exposed to environmental tobacco smoke (ETS) under real-life conditions using the urinary NNK metabolites 4-(N-methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) and [4-(N-methylnitrosamino)-1-(3-pyridyl)but-1-yl]-beta-O-D-glucosiduronic acid (NNAL-Gluc). NNK was not detectable (&lt;0.5 ng m^-3) in 11 rooms in which smoking did not occur. The mean NNK concentration in 19 rooms in which smoking took place was 17.5 (2.4-50.0) ng m^-3. The NNK levels significantly correlated with the nicotine levels (r=0.856; p&lt; 0.0001). Of the 29 non-smokers investigated, 12 exhibited no detectable NNAL and NNAL-Gluc excretion (&lt;3 pmol day) in their urine. The mean urinary excretion of NNAL and NNAL-Gluc of the 17 remaining non-smokers was 20.3 (&lt;3-63.2) and 22.9 (&lt;3-90.0) pmol day^-1, respectively. Total NNAL excretion (NNAL+NNAL-Gluc) in all non-smokers investigated significantly correlated with the amount of nicotine on personal samplers worn during the week prior to urine collection (r=0.88; &lt;0.0001) and with the urinary cotinine levels (r=0.40; p=0.038). No correlation was found between NNAL excretion and the reported extent of ETS exposure. Average total NNAL excretion in the non-smokers with detectable NNAL levels was 74 times less than in 20 smokers who were also investigated. The cotinine/total NNAL ratios in urine of smokers (9900) and non-smokers (9300) were similar. This appears to be at variance with the ratios of the corresponding precursors (nicotine/NNK) in mainstream smoke (16400) and ETS (1000). Possible reasons for this discrepancy are discussed. The possible role of NNK as a lung carcinogen in non-smokers is unclear, especially since NNK exposure in non-smokers is several orders of magnitude lower than the ordinary exposure to exogenous and endogenous N-nitrosamines and the role of NNK as a human lung carcinogen is not fully understood.     

Chromosome aberrations among cigarette smokers in Colombia

Worldwide, the annual morbimortality caused by cigarette smoking is a major public health concern. In Colombia, up to 33% of the adult population has smoked at some point in life, raising important national issues on the disease burden from tobacco. The aim of this study was to establish whether cigarette smoking increases the frequency of chromosome aberrations (CA) in peripheral blood lymphocytes of smokers (n = 52) compared with non-smokers (n = 52) in Popayán, Colombia. After signing a consent form, volunteers provided a blood sample (20 ml) to establish cell cultures at 52 h. For CA analysis, 100 complete metaphase cells from each subject were evaluated. The CA frequency was significantly higher in smokers (8.38 +/- 0.61) than in non-smokers (3.13 +/- 0.29), showing the highest number of CA (14.83 +/- 1.01) among heavy smokers (>20 pack-years). Interestingly, light smokers (< or =10 pack-years) also showed a significant increase in CA when compared to non-smokers (6.62 +/- 0.53 versus 3.13 +/- 0.29, P < 0.01, respectively). In addition, a significant positive correlation was found between the frequency of CA and the intensity of smoking in pack-years (R2 = 0.60). Our study indicates that the genotoxic effects in lymphocytes from smokers are most likely caused by cigarette smoke constituents, providing scientific evidence to encourage national campaigns to prevent tobacco consumption.