COPD increases the risk of squamous histological subtype in smokers who develop non-small cell lung carcinoma.

BACKGROUND: Squamous cell carcinoma has a stronger association with tobacco smoking than other non-small cell lung cancers (NSCLC). A study was undertaken to determine whether chronic obstructive pulmonary disease (COPD) is a risk factor for the squamous cell carcinoma histological subtype in smokers with surgically resectable NSCLC. METHODS: Using a case-control design, subjects with a surgically confirmed diagnosis of squamous cell carcinoma were enrolled from smokers undergoing lung resection for NSCLC in the District Hospital of Ferrara, Italy. Control subjects were smokers who underwent lung resection for NSCLC in the same hospital and had a surgically confirmed diagnosis of NSCLC of any histological type other than squamous cell. RESULTS: Eighty six cases and 54 controls (mainly adenocarcinoma, n = 50) were enrolled. The presence of COPD was found to increase the risk for the squamous cell histological subtype by more than four times. Conversely, the presence of chronic bronchitis was found to decrease the risk for this histological subtype by more than four times. Among patients with chronic bronchitis (n = 77), those with COPD had a 3.5 times higher risk of having the squamous cell histological subtype. CONCLUSIONS: These data suggest that, among smokers with surgically resectable NSCLC, COPD is a risk factor for the squamous cell histological subtype and chronic bronchitis, particularly when not associated with COPD, is a risk factor for the adenocarcinoma histological subtype.

A randomized study comparing two different schedules of administration of cisplatin in combination with gemcitabine in advanced nonsmall cell lung carcinoma.

BACKGROUND: This randomized trial was designed to investigate the feasibility, toxicity, and activity of two different schedules of gemcitabine plus cisplatin in previously untreated patients with advanced (International Union Against Cancer (UICC) Stage IIIB-IV) nonsmall cell lung carcinoma (NSCLC). METHODS: From February 1997 to September 1998, 82 patients with advanced NSCLC were entered onto the study and were randomized to gemcitabine 1000 mg/m(2) on Days 1, 8, and 15 plus cisplatin 80 mg/m(2) on Day 2 (arm A) or Day 15 (arm B) every 28 days. RESULTS: All the patients were assessable for toxicity (arm A/arm B: 151/177 cycles; median, 4 of 5 cycles per patient), and the following Grade 3-4 toxicities were reported (percentage of cycles in arm A vs. arm B): anemia, 7.9% and 2.3% (P < 0.05); leukopenia, 6.0% and 6.7%; thrombocytopenia, 15.0% and 1.6% (P < 0.01); no World Health Organization (WHO) Grade 3-4 nonhematologic toxicities were observed. These side effects led to gemcitabine dose reductions in 35.1% of courses in arm A and 22.0% of courses in arm B (P < 0.05) and to gemcitabine omissions in 28.5% of courses in arm A versus 7.3% of courses in arm B (P < 0.01). Dose intensities (DIs) of gemcitabine were 607.5 mg/m(2)/week in arm A and 711.6 mg/m(2)/week in arm B (P < 0.01); DIs of cisplatin were 18. 1 mg/m(2)/week in arm A and 18.8 mg/m(2)/week in arm B. The total delivered doses of gemcitabine were 9315.5 mg/m(2) in arm A and 12, 631.0 mg/m(2) in arm B (P < 0.01); the total delivered doses of cisplatin were 277.1 mg/m(2) in arm A and 333.0 mg/m(2) in arm B (P < 0.01). Response rates according to intention to treat were 40.4% (95% confidence interval [CI], 25.5-55.3) in arm A and 45% (95% CI, 29.5-60.5) in arm B. The overall median duration of response was 7.4 months; the median time to disease progression was 6 months (95% CI, 3-9) in arm A and 9 months (95% CI, 4-14) in arm B (P < 0.02); the median overall survival was 10 months (95% CI, 7.0-12.5) in arm A and 17 months (95% CI, 13.0-21.6) in arm B (P < 0.01); the 1-year survival rates were 34% and 63%, respectively. CONCLUSIONS: Our data show that arm B (cisplatin on Day 15) is less toxic than arm A (cisplatin on Day 2) and allows the administration of significantly higher total doses and dose intensities of chemotherapy. No significant differences in response rates were observed between the two schedules; patients on arm B experienced a significantly more prolonged progression free and overall survival; however, the study was not powered to detect differences in these outcomes.

Gemcitabine monotherapy in elderly patients with advanced non-small cell lung cancer: a multicenter phase II study.

BACKGROUND: This trial investigated the activity and toxicity of gemcitabine in previously untreated elderly (> 70 years) patients with advanced (stage IIIB-IV) non-small cell lung cancer (NSCLC). PATIENTS AND METHODS: From January 1997 to July 1998, 46 patients with advanced NSCLC aged over 70 years with a performance status of 0-2 were entered into the study. Gemcitabine 1000 mg/m2 was administered as a 30-min infusion once a week for 3 weeks followed by a week of rest; cycles were repeated every 4 weeks. RESULTS: Forty-four patients were evaluable for response. One complete response and nine partial responses were observed, for an overall response rate of 22.2% (95% C.I.: 11.3-37.5). The median time to disease progression was 5.1 months (95% C.I.: 3.5-6.7), the median duration of response was 6.3 months, and the median overall survival time 6.75 months (95% C.I.: 5.3-8.2). All patients were evaluable for toxicity (184 cycles, median = 3 cycles/patient) and no grade 4 hematologic toxicities were reported. WHO grade 3 leukopenia, neutropenia and anemia occurred in 3.3, 0.5 and 1.1% of cycles, respectively. Grade 3 skin rash occurred in 4.3% of patients. These side effects led to treatment discontinuation in two patients. CONCLUSION: Our data show that gemcitabine is active and well tolerated in patients aged over 70 years with advanced NSCLC.

Effects of nicotine replacement therapy on markers of oxidative stress in cigarette smokers enrolled in a smoking cessation program.

Twenty healthy, asymptomatic long-term cigarette smokers (8 males, 12 females; mean age: 43 +/- 9 years) were selected at random from a larger series receiving nicotine replacement therapy (NRT) for 12 weeks to study the effects of NRT on plasma markers of oxidative stress. Plasma aliquots, obtained at baseline (T0) and after 12 weeks (T12) of NRT, were used to measure malondialdeyde (MDA) and total Trolox-equivalent antioxidant capacity (TEAC). In subjects who completely quit smoking ('quitters', n = 10), MDA was higher at T0 (1.08 mumol/l, interquartile range 0.85-1.16) than at T12 (0.71 mumol/l, range 0.32-0.92; p < 0.01), and TEAC was lower at T0 (1.20 mM, range 1.11-1.31) than at T12 (1.43 mM, range 1.31-1.49; p < 0.05). In subjects who had only reduced the number of cigarettes smoked per day ('reducers', n = 10), differences between the T0 and T12 levels of MDA (0.81 [0.75-0.96] vs. 0.76 [0.58-0.84] mumol/l) and TEAC (1.28 [1.05-1.50] vs. 1.25 [1.09-1.42] mM) were not significant. At T0, MDA and cotinine levels correlated in reducers (r = 0.79, p < 0.05) and, though not significantly, in quitters (r = 0.50, p = 0.12). At T12 this relationship between MDA and cotinine was still present in the reducers (r = 0.70, p < 0.05), while the scatter of points in quitters was completely dispersed (r = (0.09). These results show that smoking cessation but not smoking reduction is associated with decreased markers of oxidative stress in the plasma of active cigarette smokers.

ICAM-1-independent adhesion of neutrophils to phorbol ester-stimulated human airway epithelial cells.

Intercellular adhesion molecule-1 (ICAM-1) is the only inducible adhesion receptor for neutrophils identified in bronchial epithelial cells. We stimulated human airway epithelial cells with various agonists to evaluate whether ICAM-1-independent adhesion mechanisms could be elicited. Phorbol 12-myristate 13-acetate (PMA) stimulation of cells of the alveolar cell line A549 caused a rapid, significant increase in neutrophil adhesion from 11 +/- 3 to 49 +/- 7% (SE). A significant increase from 17 +/- 4 to 39 +/- 6% was also observed for neutrophil adhesion to PMA-stimulated human bronchial epithelial cells in primary culture. Although ICAM-1 expression was upregulated by PMA at late time points, it was not affected at 10 min when neutrophil adhesion was already clearly enhanced. Antibodies to ICAM-1 had no effect on neutrophil adhesion. In contrast, antibodies to the leukocyte integrin beta-chain CD18 totally inhibited the adhesion of neutrophils to PMA-stimulated epithelial cells. These results demonstrate that PMA stimulation of human airway epithelial cells causes an increase in neutrophil adhesion that is not dependent on ICAM-1 upregulation.

Serum antibodies to benzo(a)pyrene diol epoxide-DNA adducts in the general population: effects of air pollution, tobacco smoking, and family history of lung diseases.

Polycyclic aromatic hydrocarbons (PAHs) are almost ubiquitous pollutants that may interact with metabolic systems in human tissues and eventually cause cancer. PAH-adducted DNA becomes antigenic and antibodies anti-benzo(a)pyrene diol epoxide (BPDE)-DNA may be found in serum of PAH-exposed subjects. The presence of serum antibodies anti-BPDE-DNA adduct was investigated in 1345 individuals from family clusters of the general population of a small area in central Italy in whom information about smoking habits, site of residence, and personal and family history of lung diseases, including cancer, were obtained. Anti-BPDE-DNA antibodies in the sera were detected with a direct ELISA and the association of anti-BPDE-DNA antibodies with subjects' data from a standardized respiratory questionnaire including age, occupation, tobacco smoking habits, respiratory symptoms, and family history of respiratory diseases was subsequently tested by multivariate logistic regression analysis. The overall prevalence of subjects with anti-BPDE-DNA antibodies was 21.0% (n=283), with no differences between males and females. Anti-BPDE-DNA positivity was associated with living in the urban area [odds ratio (OR), 1.49; 95% confidence interval (CI), 1.16-1.92], with active tobacco smoking (OR, 1.25; 95% CI, 1.06-1.48), and with family history of lung cancer (OR, 1.30; 95% CI, 0.90-1.88), and positivity increased with the number of members in the family cluster positive to anti-BPDE-DNA antibodies (OR, 1.30; 95% CI, 1.03-1.65). This study on a large general population sample indicates that serum anti-BPDE-DNA antibodies may be considered as biomarkers of exposure to environmental carcinogens and of DNA damage. The genetic and familial components of their association with tobacco smoking lend further support to the argument about the familial predisposition to lung cancer.

Plasma 3-nitrotyrosine in cigarette smokers.

Peroxynitrite has been associated with increased oxidative reactions and DNA damage in inflamed tissues as it may cause a reduction of plasma antioxidants as well. Nitration of tyrosine residues of proteins leads to the production of 3-nitrotyrosine (NTYR), which may be considered as a marker of NO.-dependent oxidative damage. We developed a highly sensitive method to detect NTYR in human plasma and tested it in cigarette smokers and in healthy control subjects. Peripheral venous blood (10 ml) was obtained in 20 healthy, asymptomatic cigarette smokers (13 males, 7 females; age: 49 +/- 11 yr) and in 18 healthy nonsmokers (10 males and 8 females; age: 36 +/- 6 yr). In smokers, plasma nicotine, cotinine, and expired CO levels were measured. NTYR was determined with a sequential HPLC/gas chromatography-thermal energy analysis (GC-TEA) technique. The total plasma Trolox-equivalent antioxidant capacity (TEAC) was also measured using metmyoglobin as peroxidase and a phenothiazine as a radical donor. NTYR was detectable (detection limit: 0.02 ng/injection) in 11 smokers (mean +/- SD: 1.60 +/- 1.24 ng/mg protein) and in two nonsmokers (1.10 and 1.20 ng/mg protein, respectively). NTYR was not associated with nicotine and cotinine levels or expired CO in smokers. Plasma TEAC in smokers was significantly lower (0.43 +/- 0.38 mM) than in nonsmokers (1.42 +/- 0.3 mM; p < 0.001) and showed a biphasic, negative relationship with NTYR (r = 0.96, p < 0.001). This highly sensitive HPLC/GC-TEA method for detection and quantitation of plasma NTYR may be used for monitoring oxidative reactions associated with tobacco smoking. This assay might be incorporated into molecular epidemiologic studies for lung chronic inflammatory and neoplastic disorders in which exposure to oxidants may be an important risk factor.

Presence and persistence of serum anti-benzo[a]pyrene diolepoxide-DNA adduct antibodies in smokers: effects of smoking reduction and cessation.

Among biomarkers of tobacco smoke (TS)-induced genotoxic damage, benzo[a]pyrene diolepoxide-DNA adducts (BPDE-DNA) are extensively studied. Adducted DNA becomes antigenic and antibodies anti-BPDE-DNA (BPDE-DNA-Abs) may be found in serum of exposed subjects. Little is known about the persistence of BPDE-DNA, and no study has been performed to evaluate the persistence of BPDE-DNA-Abs after cessation of exposure. Fifty heavy smokers, enrolled in a smoking cessation program with nicotine patch substitution therapy, were evaluated for the presence of BPDE-DNA-Abs before (w0) and 1, 3, 6 and 12 weeks (w1-12) after the start of the program. Nicotine or placebo patches were randomly assigned to the subjects. BPDE-DNA-Abs were determined in serum by non-competitive ELISA. After the start of the cessation program, 28 subjects quit smoking (group Q) and the other 22 reduced by about 75% the number of cigarettes smoked per day (group R). At the start of the program (w0) 8% of subjects were positive. At w1 the prevalence of positivity had increased both in subjects who quit smoking (Q: 21%) and in subjects who had reduced the number of cigarettes per day (R: 27%). Positivity remained stable up to w12 (21%) for group Q, whereas it increased to 41% in group R. Serum BPDE-DNA-Abs can be detected in smokers, and their persistence for months after smoking cessation suggests their usefulness for relatively long-term surveys. The low percentage of positivity in actual heavy smokers and the increase in antibody positivity with smoking cessation or reduction must be taken into account when interpreting serum BPDE-DNA-Ab measurement in exposed individuals.

Detection of N7-methyldeoxyguanosine adducts in human pulmonary alveolar cells.

Alkylating agents may cause DNA damage in different human cells and tissues, including lungs. For instance, tobacco-specific N-nitrosamines are known to produce methyl-DNA adducts, such as N7-methyldeoxyguanosine, and to induce lung tumors. We applied a combined high-performance liquid chromatography (HPLC)/32P-postlabeling technique for measurement of N7-methyldeoxyguanosine in human pulmonary alveolar cells (HPAC). Thirty patients (13 males, 17 females; mean age 51 +/- 17 yr) undergoing bronchoalveolar lavage for diagnosis of nonmalignant lung diseases were studied. DNA was extracted from HPAC, digested to 2'-deoxyribonucleotide 3'-monophosphates and HPLC separated to obtain deoxyguanosine (dGp) and N7-methyldeoxyguanosine (N7-MedGp) monophosphates. Fractions corresponding to normal (1:10,000) and N7-methylated dGp were subsequently 32P-postlabeled by T4 polynucleotide kinase with high specific activity 32P-ATP, resolved by two-dimensional thin-layer chromatography (TLC) and autoradiographed after 3 to 18 h exposure. Spots corresponding to dGp and N7-MedGp were scraped off the plates and quantitated by liquid scintillation counting to calculate direct molar ratios. Recovered HPAC (14.4 +/- 10.0 x 10(6)) were predominantly macrophages (73.8 +/- 16.4%) and lymphocytes (9.8 +/- 11.6%). N7-MedGp was detected in 11 patients, the level ranging from 0.10 to 48.03 fmol/micrograms DNA which corresponded to 0.31-79.00 x 10(-6) N7-MedGp/dGp ratios. Detection of N7-MedGp in HPAC was associated with the smoking habit of patients: N7-MedGp was present in 7 of 10 smokers, 2 of 10 ex-smokers, and 2 of 10 nonsmokers (P < 0.05). These results show that HPAC may be used for molecular dosimetry of DNA damage by alkylating agents, including tobacco-specific N-nitrosamines, in cigarette smokers and thus used for cancer risk assessment.

Host factors in lung carcinogenesis.

The interaction between aetiological factors (e.g. tobacco smoke) and target cells in lung tissues is modulated by host factors, leading to variable risk among individuals. We have studied some of the metabolic properties of the human lung and the effects on them of tobacco smoke. The activity of activating enzymes is induced by recent tobacco smoke exposure, whereas that of inactivating enzymes is depressed, and this effect is greater in patients with lung cancer than in controls. This imbalance between activating and detoxifying enzymes in the lung may be a key factor in determining the genetic damage to the lung. These differences between lung cancer patients and controls in metabolic activities in lung tissues may be documented at a phenotypic level as well as at a genotypic level. Also wide interindividual differences have been shown in deoxyribonucleic acid (DNA) repair enzymes and in the extent of DNA damage.

7-Alkyldeoxyguanosine adduct detection by two-step HPLC and the 32P-postlabeling assay.

7-Alkyldeoxyguanosine DNA adducts may be a marker for some N-nitroso compound exposures and subsequent human cancer risk. A sensitive and highly specific assay for the detection of 7-methyl-2'-deoxyguanosine-3'-monophosphate (7-methyldGp) and 7-ethyl-2'-deoxyguanosine-3'-monophosphate (7-ethyldGp) has been developed by combining two different HPLC purification steps with the 32P-postlabeling assay. We previously reported that ion-pair reverse-phase (IP) chromatography coupled with the 32P-postlabeling assay detects 7-methyldGp in human lung, but have found that other nucleotides and unknown adducts co-elute. Thus, weak anion exchange (AE) HPLC was added in tandem with IP HPLC prior to the 32P-postlabeling assay. 2'-Deoxyguanosine-3'-monophosphate (dGp) is incorporated into the assay as an internal standard for the assessment of enzyme labeling efficiency and adduct recovery. The methodology was validated using radiolabeled DNA and liquid scintillation counting, which accounts for adduct loss from enzymatic digestion to detection. Levels of 7-ethyldGp also were correlated with accelerator mass spectrometry. The overall adduct recovery with this method was 58% for 7-methyldGp and 98% for 7-ethyldGp. The detection limit for both assays using 100 micrograms of DNA was one adduct in 10(8) unmodified dGp. 7-MethyldGp and 7-ethyldGp levels were determined in ten human lung samples at levels of 1.4-5.4 and 0.6-3.1 adducts per 10(7) dGp respectively, and in five human lymphocyte samples at levels of 5.0-8.3 and 0.3-1.4 adducts per 10(7) dGp respectively. Combining the two HPLC purification steps and the 32P-postlabeling assay attains chemical specificity, retains sufficient quantitative sensitivity and should be useful in human biomonitoring studies.

Standardization of the 32P-postlabeling assay for polycyclic aromatic hydrocarbon-DNA adducts.

A modified method for the 32P-postlabeling assay that permits standardized quantitation for specific polycyclic aromatic hydrocarbon (PAH)-DNA adducts is described. This method has been designed to test the components of the 32P-postlabeling assay for use in the chemically specific detection of individual adducts with the ultimate goal of testing the biological significance of PAH-DNA adducts in humans. The approach relies upon high performance liquid chromatography (HPLC), concomitant labeling of 2'-deoxyguanosine (dGp) as an internal standard and thin layer chromatography (TLC), which identifies unmodified nucleotides along with PAH-DNA adducts on the same TLC plate. This method assesses labeling efficiency, detects the presence of unknown inhibitors, assesses the adequacy of digestion (when combined with HPLC) and allows for the development of calibration curves for directly determined molar ratios (adduct:internal standard). Chemically synthesized adduct standards and quantitative 32P-postlabeling data have been corroborated by UV spectroscopy, fluorescence spectroscopy and liquid scintillation counting where radiolabeled materials were available. Labeling efficiencies of the PAH-DNA adducts were found to be up to 100-fold less than expected and depended upon both the adduct and adduct levels (lower levels being less efficiently detected). The presence of unmodified nucleotides resulted in a 1.5-fold lower labeling efficiency. Mixtures of selected PAH-DNA adducts did not affect the labeling efficiencies of each other. The data suggest that previous 32P-postlabeling assay studies for PAH-DNA adducts may have underestimated adduct levels due to variations in labeling efficiency.

Combined micropreparative techniques with synchronous fluorescence spectroscopy or 32P-postlabelling assay for carcinogen-DNA adduct determination.

Methods for the detection of carcinogen-DNA adducts require both sensitivity and specificity. The 32P-postlabelling assay is highly sensitive but lacks adduct specificity. The strategy reported herein combines micropreparative techniques including HPLC and immunoaffinity chromatography (IAC) to enhance chemical specificity. The resultant assays have retained sensitivity for human DNA analysis. Combined HPLC and 32P-postlabelling has detected 7-methyldeoxyguanosine in human lung samples, while combined IAC and 32P-postlabelling has detected O6-methyldeoxyguanosine adducts in stomach tissue. The limits of detection are one adduct in 10(7) and 10(8) unmodified deoxyguanosine (dG), respectively. IAC was combined with 32P-postlabelling to detect polycyclic aromatic hydrocarbon (PAH)-DNA adducts in human lung. The detection limit was one adduct in 10(7) dG. Our laboratory has also employed synchronous fluorescence spectroscopy (SFS) for the detection of adducts formed from benzo[a]pyrene in human lung. Complex fluorescence matrices suggest the presence of other PAH-DNA adducts. Both the SFS assay and the 32P-postlabelling assay were applied to a series of human samples and a high correlation was found for adduct levels. The development of such assays using synthetic standards, internal standards, determination of calibration curves and validation with corroborating methods is required for chemically specific and sensitive adduct detection.

Polycyclic aromatic hydrocarbon-DNA adducts and the CYP1A1 restriction fragment length polymorphism.

Human cancer risk assessment at a genetic level involves the investigation of carcinogen metabolism and DNA adduct formation. Wide interindividual differences in metabolism result in different DNA adduct levels. For this and other reasons, many laboratories have considered DNA adducts to be a measure of the biologically effective dose of a carcinogen. Techniques for studying DNA adducts using chemically specific assays are becoming available. A modification of the 32P-postlabeling assay for polycyclic aromatic hydrocarbon DNA adducts described here provides potential improvements in quantification. DNA adducts, however, reflect only recent exposure to carcinogens; in contrast, genetic testing for metabolic capacity indicates the extent to which carcinogens can be activated and exert genotoxic effects. Such studies may reflect both separate and integrated risk factors together with DNA adduct levels. A recently described restriction fragment length polymorphism for the CYP1A1, which codes for the cytochrome P450 enzyme primarily responsible for the metabolic activation of carcinogenic polycyclic aromatic hydrocarbons, has been found to be associated with lung cancer risk in a Japanese population. In a subset of individuals enrolled in a U.S. lung cancer case-control study, no association with lung cancer was found.

Carcinogen metabolism in human lung tissues and the effect of tobacco smoking: results from a case--control multicenter study on lung cancer patients.

Cigarette smoking is the strongest risk factor for lung cancer, but genetically determined variations in the activities of pulmonary enzyme that metabolize tobacco-derived carcinogens may affect individual risk. To investigate whether these enzymes (e.g., CYP1A-related) can serve as markers for carcinogen-DNA damage, lung tissue specimens were taken during surgery from middle-aged men with either lung cancer or non-neoplastic lung disease. Phase I [aryl hydrocarbon hydroxylase (AHH), ethoxycoumarin O-deethylase (ECOD)] and phase II (epoxide hydrolase, UDP-glucuronosyltransferase, glutathione S-transferase) enzyme activities, glutathione and malondialdehyde contents were determined in lung parenchyma and/or bronchial tissues; some samples were also analyzed for DNA adducts, using 32P-postlabeling. The data were then analyzed for the following: a) differences in metabolic profiles between bronchial and parenchymal lung tissue; b) the effect of recent exposure to tobacco smoke on enzyme inducibility and benzo[a]pyrene metabolism; c) differences in enzyme inducibility between lung cancer and non-lung cancer patients; d) the effect of smoking on metabolism of mutagens in vitro; e) pulmonary DNA adduct levels and AHH activity in lung parenchyma of smokers and ex-smokers; f) lipid peroxidation products in lung tissue from lung cancer and non-lung cancer patients, as related to smoking habits and degree of airway obstruction; and g) prognostic value of AHH pulmonary activity in lung cancer patients. The results demonstrate a pronounced effect of tobacco smoke on pulmonary metabolism of xenobiotics and prooxidant state and suggest the existence of a metabolic phenotype at higher risk for tobacco-associated lung cancer.

An improved fluorometric assay for dosimetry of benzo(a)pyrene diol-epoxide-DNA adducts in smokers' lung: comparisons with total bulky adducts and aryl hydrocarbon hydroxylase activity.

An improved high-performance liquid chromatography/fluorometric assay has been established to quantitate the benzo(a)pyrene (BP) tetrols released after acid hydrolysis of lung DNA from lung cancer patients, so that the formation of benzo(a)pyrene diol-epoxide-DNA adducts can be measured. The r-7,c-10,t-8,t-9-tetrahydroxy-7,8,9,10-tetrahydro-BP isolated by high-performance liquid chromatography was determined by chromatography in two different solvent systems and fluorescence spectroscopy. This assay has a detection limit of 2 pg of r-7,c-10,t-8,t-9-tetrahydroxy- 7,8,9,10-tetrahydro-BP, requires 100-500 micrograms of DNA, and can measure 1 adduct/10(8) unmodified nucleotides. As this assay does not use immunoaffinity chromatography or solvent extraction, it allows a > 90% recovery of benzo(a)pyrene diol-epoxide-DNA adducts. This procedure has been tested on 13 DNA samples prepared from nontumorous lung parenchyma taken from lung cancer patients at surgery and revealed the presence of DNA adducts of the anti-benzo(a)pyrene diol-epoxide in 9 of 11 samples from smokers and in 2 of 2 ex-smokers. In only two samples from smokers the formation of adducts derived from syn-benzo(a)pyrene diol-epoxide was detected. A 15-fold variation in DNA adduct level was found in 11 of 13 DNA samples, with a range of 0.6-9.9 adducts of benzo(a)pyrene diol-epoxide/10(8) nucleotides. In samples containing both anti- and syn-benzo(a)pyrene diolepoxide-DNA adducts, the anti/syn adduct ratio is 2:1. A highly significant correlation was found between pulmonary microsomal aryl hydrocarbon hydroxylase activity and the level of benzo(a)pyrene diolepoxide-DNA adduct (r = 0.91; P < 0.001; n = 13). A crude linear correlation between the amounts of these adducts and those of bulky DNA adducts determined by 32P-postlabeling assay was observed in the same samples (r = 0.78; P < 0.02; n = 13). Thus this highly sensitive and specific procedure is suitable for measuring benzo(a)pyrene diolepoxide-DNA adducts in human tissues from environmentally exposed subjects and could be adapted to measure polycyclic aromatic hydrocarbons other than BP.

Cigarette smoke inhibits cytosolic but not microsomal epoxide hydrolase of human lung.

The effect of cigarette smoke exposure on the activity of cytosolic and microsomal epoxide hydrolase (EH) has been investigated in human lung. Patients were classified as 'recent smokers' (n = 9) or 'non-recent smokers' (n = 10) according to whether they were or were not still smoking 1 month before surgery. Cytosolic EH was measured with [3H]trans-stilbene oxide as a substrate, whereas microsomal EH was measured with [7-3H]styrene oxide as a substrate. Microsomal EH activity did not differ between recent smokers (2.51 +/- 0.93 nmol min-1 mg-1) and non-recent smokers (2.74 +/- 1.10 nmol min-1 mg-1), whereas cytosolic EH activity was significantly lower in recent smokers (6.46 +/- 1.79 pmol min-1 mg-1) than in non-recent smokers (8.41 +/- 2.09 pmol min-1 mg-1, P less than 0.05). Cytosolic EH activity was correlated with the number of days that had passed since the cessation of smoking (r = 0.58, P less than 0.05) and the effect was dose-dependent, since the enzyme activity was inversely correlated with the number of cigarettes smoked per day (r = 0.85, P less than 0.01). This suggests that recent smoking exposure inhibits the activity of cytosolic EH but not microsomal EH, and that the inhibition increases with the number of cigarettes smoked per day. The contribution of cytosolic enzymes to xenobiotic metabolism may be remarkable in extrahepatic tissues. The inhibition of cytosolic EH by tobacco smoke may reduce the inactivation of carcinogenic epoxides in human lung tissues and so may increase a person's susceptibility to lung cancer.

Carcinogen metabolism and DNA adducts in human lung tissues as affected by tobacco smoking or metabolic phenotype: a case-control study on lung cancer patients.

Individual variations in activity of pulmonary enzymes that metabolize tobacco-derived carcinogens may affect an individual's cancer risk from cigarette smoking. To investigate whether some of these enzymes (e.g., cytochrome P450IA-related) can serve as markers for carcinogen-induced DNA damage accumulating in the lungs of smokers, non-tumorous lung tissue specimens were taken during surgery from middle-aged men with either lung cancer (n = 54) or non-neoplastic lung disease (n = 20). Phase I (AHH, ECDE) and phase II (EH, UDPGT, GST) enzyme activities, glutathione and malondialdehyde contents were determined in lung parenchyma and/or bronchial tissues; some samples were analyzed for DNA adducts, using 32P-postlabeling. Data analysis of subsets or the whole group of patients yielded the following results. (1) Phase I and II drug-metabolizing enzyme (AHH, EH, UDPGT, GST) activities in histologically normal surgical specimens of lung parenchyma were correlated with the respective enzyme activities in bronchial tissues of the same subject. (2) In lung parenchyma, enzyme (AHH, ECDE, EH, UDPGT) activities were significantly and positively related to each other, implying a similar regulatory control of their expression. (3) Mean activities of pulmonary enzymes (AHH, ECDE) were significantly (2- and 7-fold, respectively) higher in lung cancer patients who had smoked within 30 days before surgery (except GST, which was depressed) than in cancer-free subjects with a similar smoking history. (4) In the cancer patients, the time required for AHH, EH and UDPGT activities to return to the level found in non-smoking subjects was several weeks. (5) Bronchial tree and peripheral lung parenchyma preparations exhibited a poor efficiency in activating promutagens to bacterial mutagens in Salmonella. However, they decreased the mutagenicity of several direct-acting mutagens, an effect which was more pronounced in tissue from recent smokers. GSH concentration and GST activity were positively correlated with mutagen inactivation in the same sample. (6) In recent smokers, AHH activity in lung parenchyma was positively correlated with the level of tobacco smoke-derived DNA adducts. (7) Pulmonary AHH and EH activity had prognostic value in tobacco-related lung cancer patients. (8) An enhanced level of pro-oxidant state in the lungs was associated with recent cigarette smoking. Malondialdehyde level in lung parenchyma was associated with the degree of small airway obstruction, suggesting a common free radical-mediated pathway for both lung cancer induction and small airway obstruction.(ABSTRACT TRUNCATED AT 250 WORDS)

Comparison of pulmonary DNA adduct levels, measured by 32P-postlabelling and aryl hydrocarbon hydroxylase activity in lung parenchyma of smokers and ex-smokers.

In order to compare pulmonary DNA adducts and aryl hydrocarbon hydroxylase (AHH) activity, we have measured these two parameters in non-neoplastic surgical lung parenchymal samples from four ex-smokers and 19 smokers, out of 20 patients operated for lung cancer, and three for nonmalignant lung diseases. DNA adducts were determined by scintillation counting after 32P-postlabelling analysis. The microsomal fractions of the same lung specimen were assayed for AHH activity by a fluorometric method. Autoradiograms of DNA adducts found in lungs of smokers revealed two distinct diagonal radioactive zones that were absent in ex-smokers. The smokers had significantly higher levels (1.68-13.4 DNA adducts/10(8) nucleotides; mean +/- SD 5.38 +/- 3.19) than ex-smokers (0.23-2.21; 1.09 +/- 0.84). AHH activity in smokers ranged from 0.01 to 0.69 pmol/min/mg. This activity was significantly (P less than 0.05) higher in smokers (0.26 +/- 0.26) who had smoked until 1 week before surgery than in those who had stopped smoking for greater than 7 days (0.11 +/- 0.11). A positive linear correlation between DNA adduct levels and AHH activity (r = 0.69; P less than 0.001; n = 19) was found in smokers. This relationship could explain why AHH inducibility appears to be a crude marker for lung cancer risk in smokers.

Reversibility of complete unperfusion in a patient with recurrent hemoptysis.

We present a case of a 68-year-old woman with a history of mild smoking and chronic bronchitis who showed recurrent hemoptysis. She presented with a nearly normal chest roentgenogram, a non-diagnostic fiberoptic bronchoscopy and a computed tomography and lung scanning both of which were highly suggestive for malignancy. In fact, the former showed obstruction of the main left bronchus, of the superior bronchus for the left upper lobe and of the apical bronchus for the left lower lobe, the latter showed a total cessation of blood flow through the left lung. Pulmonary angiography, however, was normal and aortography showed dilatated and twisted left bronchial arteries. Computed tomography and lung scanning came back to normal after bronchoscopic aspiration of endobronchial clots and a nonspecific antibiotic therapy were carried out. Although very infrequent, bronchial stenosis on CT and complete monolateral unperfusion on lung scintigraphy may occur in patients with hemoptysis of benign origin. We recommend the use of pulmonary arteriography in patients with the above pattern when diagnostic doubt remains after bronchoscopy.