Pulmonary Lipid Peroxidation in Cigarette Smokers and Lung Cancer Patients

Pulmonary Lipid Peroxidation in Cigarette Smokers and Lung Cancer Patients* Stefano Petruzzelli, M.D., Ph.D.; E'no Hietanen, M.D.; Helmut Bartsch, Ph.D.; Anne Marie Camus, B.S.; AlfredQ Mussi, M.D.; Carlo Alberto Angeletti, M.D.,. RG.G.P.; Rodolfo Saracci, M.D.; and Carlo Giuntini, M.D., RG.G.E

Lipid peroxidation (LPO) was studied in lung tissues of patients with lung cancer (LC, n=37) or DOnlung cancer (NLC, n = 13) and its relationships with the smoking habits and the degree of airway obstruction were investigated. Specimens of peripheral lung parenchyma, free of tumor tissue, were taken and the malondialdehyde (MDA) content was measured in the S-12 fractions. Airway obstruction was assessed by Bow-volume curves, and data were expressed as percentage of the predicted values. Cigarettes smoked were expressed as pack-years. The patients with LC and NLC did not differ by MDA content, age, and number of pack-years. On the contrary, FEF75-85 and MEF7S were significantly lower in LC than in NLC patients (p
smokers, MDA content was higher in LC patients (0.144±0.008 Jl-mollg of tissue) than in NLC patients (0.113±0.014 mmollg tissue, p=0.059). When patients were divided into "high MDA" and '1ow MDA" groups, MEF7S was much lower in the high MDA group (35.1 ±3.4 percent) than in the low MDA group (55.1 ±8.1 percent) (p
smoking is strongly associated with enC igarette hanced risk of lung cancer (LC), but host factors

ogens of tobacco smoke, may give origin to free radical derivatives." Relative lack of oxygen may predispose organs to the burst of free radicals" and antioxidants may prevent oxidative damage to lung tissues. Ui.17 Lipid peroxidation products and reactive oxygen species have been found to be very active in binding to DNA, to cause mutations and to initiate cancers}8-9J> They are also involved in the mechanisms of myocardial ischemia, inHammatory and rheumatic diseases, and some other diseases, eg, diabetes, in man. 21-24 However, studies concerning the measurement of pulmonary LPO in relationship to the presence of LC and/or lung function impairment have, to our knowledge, not been performed to date. In previous studies, we observed increased malondialdehyde (MDA) levels in LC patients who have smoked recently (reported as preliminary results by Petruzzelli et al"), The present study was designed to examine the relationship between LPO and reduced glutathione (GSH) in lung tissues from both 1£ and nonlung cancer (NLC) patients as related to the smoking habits and to the degree of airway obstruction.

1

may also play an important role." The actual carcinogens have not been identified, even though polycyclic aromatic hydrocarbons and tobacco-specific nitrose compounds have been indicted. 3,4 Cigarette smoke alters the lung metabolism of many endogenous compounds both in experimental animals and in man,5.6 as well as the activities of many biotransforming enzymes in lung tissues.?" Cigarette smoke is a known source of oxidants," and the formation of free radicals and consequent lipid peroxidation (LPO) have been associated with chronic obstructive pulmonary disease (COPD)IO and lung cancer," Also antioxidant deficiency may be related to COPD and lung cancer}2,13 Benzo(a)pyrene, one of the most representative carcin*From the CNR Institute of Clinical Physiology, Second Medical Clinic, and Section of Thoracic Surgery, University of Pisa, Italy, Unit of Environmental Carcinogens and Host Factors, and Unit of Analytical Epidemiology, International Agency for Research on Cancer, Lyon, France. . Supported by the CNR of Italy Special Project "Oncology;" contract 87.01309.44 and by the CNR Cardiorespiratory National Group, contract 87.01479.04. Manuscript received December 18, revision accepted March 6. Reprint requests: Dr. Giuntini, Fisiopatologia Respiratoria, Clinica Medica II, Universita di Pisa, Vta Roma 67, 56126 IVa, Italy 930

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LPO lipid peroxidation; LC lung cancer; NLC nonlung cancer; MDA = malondialdehyde; GSH = glutathione

PATIENTS AND METHODS

Subjects

Fifty randomly selected male patients (mean age ± SEM, Pulmonary Lipid Peroxidation in Cigar8tte Smokers(Petruzzelli et 81)

53.0± 1.2 years) operated on for LC (n =37) or nonmalignant lung disease (n = 13) were included in the study (Table 1). None of the patients used any enzyme-inducer drug or received any radiation treatment or chemotherapy prior to surgery. Each patient, the day before surgery, completed a standardized questionnaire on diseases, work conditions, and smoking habits, as described elsewhere. R On the basis of tumor size and location and lymph node involvement, 18 cancer patients were judged as having stage I, two patients were judged as having stage II, and 15 patients were judged as having stage III lung cancer. Nonsmokers were defined as subjects who had never smoked (one patient) or who had refrained from smoking for more than six months. Airway obstruction was assessed by Bow-volume spirometry curves and data were expressed as a percentage of the predicted values derived from a general population sample in Italy.lIS 1issue Samples and Assays

TIssue specimens of peripheral lung parenchyma were obtained at surgery from each patient, and in LC patients the specimens were taken as remote as possible from the tumor in an apparently healthy part of the lung and histologically outside the tumor. Specimens were kept in ice-cold 0.15 moVL of KCI and homogenized within one to two hours after surgery, first with a blender (Polytron) and then with a Potter-Elvehjem type homogenizer in 3 ml of 0.25 moVL of sucrose, 50 mmol/L ofTris-HCI buffer, at a pH of 7.4, per gram of tissue." The homogenate was centrifuged at 12,000 g x 15 min and the resulting S-12 supernatant was frozen in liquid nitrogen and stored at - 70°C until assay. Malondialdehyde (MDA) was representative of LPO measured as thiobarbituric acid reactive substances, as described by Uchiyama and Mihara." Reduced GSH content as an index of antioxidant efficiency of lung tissues was measured according to the method of Saville" detecting nonprotein sulfhydryl compounds, which are accounted over 90 percent by reduced GSH. JR Protein content was measured according to the method of Lowry et al.29 For statistical analysis, unpaired Student's t test, regression analysis, and Xi test were used. 30 All data were expressed as mean±SEM. RESULTS

The LC patients were not significantly older Table I-Age, Smoking HiBtory, Lung Function Teats, arad Malontlialdehyde (MDA) and Glutathione (GSH) ContenD ira Lung Cancer (1£) and Nonlung Cancer (N1£) Patienta* Parameter Age, yr Smokers, % Recent smokers, % Pack-years FVC, % of predicted value FEV., % of predicted value FEF25-75, % of predicted value MEF50, % of predicted value FEF75-SS, % of predicted value MEFi5, % of predicted value MDA, JLmollg of tissue GSH, JLmollg of tissue

LC (n=37)

NLC (n=13)

53.6± 1.2 70.3

51.3±3.1 53.8 46.1 37.9±6.3 96.1 ±3.1 86.3±3.9 65.5±8.5 61.9±7.8 66.0±7.8 58.9±7.8 0.115±0.010 0.347 ± 0.055

45.9

4O.7±3.0 93.3±2.7 79.0±3.3 51.4±3.8 5O.4±4.3 46.2±3.8t 38.9±3.8t 0.129 ± 0.006 0.292±0.038

*Data are expressed as mean±SEM. "Smokers" are defined as people who have been smoking up to 6 months earlier or less; "recent smokers" are defined as people who have been smoking up to 30 days earlier or less.

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(53.6 ± 1.2 years) than NLC patients (51.3 ± 3.1 years) and also the number of pack-years was not higher in LC patients (40.7 ± 3.0) than in NLC patients (37.9±6.3) (Table 1). Among the spirometry data, the FEF75-85 (forced expiratory flow between 75 percent and 85 percent offorced vital capacity) and the MEF75 (maximal expiratory flow at 75 percent of forced vital capacity) were significantly lower in LC than in NLC patients (p<0.05, Table 1). In LC patients, the impairment of spirometry parameters was not dependent on staging of the malignant neoplasm (data not shown). As to the biochemical assays, LC and NLC patients did not differ in MDA content (0.129±O.006 vs 0.115±0.010 urnol/g of tissue) and in GSH content (0.293 ± 0.038 vs 0.347 ± 0.055 JLmoVg of tissue) (Table 1). By considering LC and NLC patients as a whole, no relationships were found between MDA and GSH contents, either taking into account the measured values (r = - 0.19) or the log-transformed values (r = - 0.16). Measured levels of MDA and GSH were not influenced by the time of storage, since no statistically significant correlation between storage time (12 ± 8 weeks) of the human lung specimens on MDA (r= -0.13) and GSH (r= -0.03) was found for all subjects or for subgroups (smokers, nonsmokers, LC patients, NLC patients). When the same patients were divided into smokers and nonsmokers (actually mainly exsmokers who had given up smoking six months earlier or more), all lung function parameters and MDA or GSH content did not differ between the two groups (Table 2). In this respect, when MDA content was related to packyears, a weak, negative correlation was found (r= -0.31, p<0.05). However, since the number of pack-years is expectedly related to the age of patients (r = 0.46, p
S (n=33)

NS (0= 17)

Age, yr Pack-years FVC, % of predicted value FEV I' % of predicted value FEF25-75, % of predicted value MEF50, % of predicted value FEF75-85, % of predicted value MEF75, % of predicted value MDA, JLmollg of tissue GSH, JLmollg of tissue

52.3± 1.4 41.7±3.5 95.8±2.3 82.7±4.2 55.8±4.2 53.3±4.3 49.1±3.9 40.5±4.3 0.128±0.006 0.308 ± 0.042

54.4±2.4 36.7±4.4 9O.6±4.4 77.2±5.5 53.5±7.3 53.7±7.9 55.5±7.4 5O.9±6.8 0.120 ± 0.009 0.302 ± 0.047

*Data are expressed as mean ± SEM. "Smokers" are defined as people who have been smoking up to 6 months earlier or less. CHEST I 98 I 4 I OCTOBER, 1990

931

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DAYS FROM STOP SMOKING FIGURE 1. Regression analysis between the malondialdehyde (MDA) content of lung tissue and the number of days since refraining from smoking before surgery in a group of patients with lung cancer (open circles) other nonmalignant lung diseases (closed circles).

patients had refrained from smoking, a significant negative log-correlation was evident (r= -0.66, p
cantly lower in the high MDA group (35.1 ± 3.4 percent of predicted value) than in the low MDA group (55.1 ± 6.3 percent of predicted value, p
The formation of free radicals and consequent LPO may be related to liver and kidney diseases," and enhanced levels ofLPO products have also been found in blood of chronic alcoholics," In the lung, the free radical production and consequent LPO has been claimed in the case of lung injury,32 paraquat poisoning,33 O2 toxicity,34 and pulmonary emphysema." Carcinogens such as benzo(a)pyrene may be activated through one-electron oxidation into diolepoxides as well as into free radical derivatives. 14 ,36,37 Moreover, free radicals are involved in the promotion phase of carcinogenesis. 11 The present study, aimed to measure LPO in human lung tissues of patients with LC or 0.4

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FIGURE 2. Comparison of malondialdehyde (MDA) and glutathione (GSH) contents between recent smokers (black bars) and the other patients (white bars). Data are expressed as means ± SEM~ statistical significances are shown by the p values (NS = p>O.05).

Pulmonary Lipid Peroxidation in CigaI8tte Smokers (Petruzzelli et 81)

Table 3-HiBtory 1JtJttJ and Lung Function Teata oflbtienta According to the Malondialdehyde (MDA) Content of Lung Tissue· Parameter

High MDA (n = 27)

LowMDA(n=23)

p value

Age, yr Diagnosis of lung cancer, % Recent smokers, % Pack-years FEF75-85, % of predicted value MEF75, % of predicted value

54.9± 1.8 85.2 63.0 35.0±3.0 47.8±4.1 35.1±3.4

51.5± 1.6 35.1 26.1 45.7±4.6 55.5±6.2 55.1±6.4

0.167 0.103 0.020 0.050 0.293 0.006

*Data are expressed as mean ± SEM. "Recent smokers" are defined as people who have been smoking up to 30 days earlier or less.

other nonmalignant lung diseases, revealed three main findings. First, LPO products, measured as MDA content of tissues, were detectable in each patient and the MDA content was not related to the antioxidant efficiency in the aqueous phase, measured as GSH content, of tissues. This result is in contrast with the observed dose-dependent protection from LPO by GSH in rat lung." However, mean GSH content in our patients (0.306±0.032 J.LmoVg of tissue) was lower than that reported in rat lung. 16 •38 Since sudden depression of GSH concentrations may be followed by a rapid restoration to normal levels, 39 measured GSH content may not reflect the actual rate of protection against LPO. The measurement of reduced GSH in the present study, performed in humans, may not be sensitive enough to detect minor changes in GSHrelated antioxidant capacity Alternative methods to measure the balance between reduced/oxidized GSH may have improved the sensitivity..fO Furthermore, MDA content in our patients had a close to normal distribution (range, 0.07 to 0.22 J.LmoVg of tissue), whereas GSH was unevenly distributed along a wider range (0.04 to 1.20 J.LmoVg of tissue) (data not shown), which may suggest a major role of other antioxidants (or other free radical trapping agents) not measured in the present study. However, GSH has been measured in the S-12 fraction which is composed, in addition to cytoplasm, also of endoplasmic reticulum where considerable proportions of MDA would stay Measurements of vitamin E, which, by the way, is much more diet dependent than GSH, is a good indicator of the antioxidant capacity in the lipid phase;" but it would not be directly pertinent to the water phase where MDA was measured. Second, cigarette smoking markedly increased pulmonary LPO and, to our knowledge, this is the first time such an effect has been demonstrated in the lung tissues of cigarette smokers. Hoidal and coworkersv-" first demonstrated an increased oxidative metabolism in alveolar macrophages of smokers. Furthermore, higher levels of MDA in plasma of smokers in respect to nonsmokers have been reported." and quite recently, cigarette smoke inhalation has been shown to increase the level of LPO in rat lung." In our study;

the cumulative smoke exposure (as expressed by the number of pack-years and adjusted by age) did not correlate with the MDA content of the lungs. More interestingly; the recent (one month or less) exposure to tobacco smoke seems to enhance markedly the MDA production in lung tissues, with a negative correlation between the MDA level and the time elapsed from the last cigarette smoked (r= -0.66). When the last 30 days of a patient's smoking habit were taken into consideration, higher MDA content was found in LC than in NLC patients. This observation, similar to that described in the case of aryl hydrocarbon hydroxylase and ethoxycoumarin O-deethylase," suggests that cigarette smoking may induce free radical reactions that might be involved in the carcinogenesis process. The higher percentage of high MDA level in 1£ patients than in NLC patients supports this hypothesis (Table 3). This suggests that in smoking 1£-prone subjects there is a high metabolic rate of cigarette smoke components and/or a lower antioxidant defense; the latter could not be verified in this study. As to the GSH, no relationships with the smoking habit of patients were found, in agreement with the reported data on GSH in the epithelial lining f1uid. 45 Third, the extent of LPO was associated with the degree of small airway obstruction. Taylor and coworkers" showed that an antioxidant de6ciency was related to abnormal FEV 1IFVC ratios. In our series, patients with high levels of MDA had signi6cantly lower MEF75, supporting experimental studies on the role of LPO as a causative agent in small airways obstruction. This observation on human lung tissues is in agreement with the results recently obtained by Richards and coworkers" showing a strong correlation between the extracellular chemiluminescence response in activated blood monocytes from smokers and the degree of small airways obstruction. Smoking may inactivate lung elastase inhibitors and cause an influx of inflammatory cells into the air spaces with the production of activated oxygen species, these being factors involved in the pathogenesis of pulmonary emphysema.P Malondialdehyde is also a byproduct of arachidonic acid cascade;" which is activated during the interaction between epithelial and in8ammatory CHEST I 98 I 4 I OCTOBER, 1990

933

cells in the lungs, and cyclooxygenase catalyzed metabolites of arachidonic acid are likely to be responsible for the ozone-induced bronchial hyperresponsiveness." This study also strengthens the usefulness of the analysis of the terminal portion of the forced expiratory curve to detect early changes due to smoking," possibly linked to the onset of an inflammatory reaction in alveolar ducts distal to the terminal bronchiole.v-" Furthermore, in the present study, LPO was associated with an enhanced cancer rate, since high MDA levels were more frequently found in LC patients than in NLC patients, independently from the smoking status of patients. Oxygen free radicals are mutagens" and the desmutagenic and anticarcinogenic effects of the antioxidant N-acetylcysteine have been shown. 52 The involvement of the prostaglandins' pathway in the activation of benzoialpyrene'" lends further support to the possible role of LPO in tobacco smoke-induced lung carcinogenesis. These findings strengthen the hypothesis, advanced on an epidemiologic basis," that interrelationships between pathogenetic factors of tobacco smoke-related diseases such as COPD and LC are likely and that efforts in the prevention of these two conditions are to be embodied in the same strategies. Free radical reactions and LPO initiated by cigarette smoke may offer a mechanism for this relationship. REFERENCES 1 fARC. fARC monographs on the evaluation of the carcinogenic risk of chemicals to humans, vol 38. Tobacco Smoking. Lyon, France, 1986 2 Gelboin H~ Carcinogens, drugs, and cytochrome P-450. N Eng) J Moo 1983; 309:105-07 3 Hoffmann D, Wynder EL. A study of tobacco carcinogenesis, XI: tumor initiators, tumor accelerators, and tumor promoting activity of condensate fractions. Cancer 1971; 27:848-64 4 Magee PN, Montesano R, Preussmann R. N-nitroso compounds and related carcinogens. In: Searle CE, ed. Chemical carcinogens, Washington, DC: ACS Monograph 1976; 173:491-624 5 Bakhle YS, Hartiala J, Toivonen H, Uotila E Effects of cigarette smoke on the metabolism of vasoactive hormones in rat isolated lungs. Br J Phannacol 1979; 65:495-99 6 Mannisto J. The effects of cigarette smoke on the fate of arachidonic acid in the rat and hamster lungs. Prostaglandin 1983; 26:681-88 7 Powell GM, Green GM. Cigarette smoking: a proposed metabolic lesion in alveolar macrophages. Biochem Phannacol 1972; 21:1785-98 8 Petruzzelli S, Camus AM, Carrozzi L, Ghelarducci L, Hindi M, Menconi GF, et al. Long-lasting effects of tobacco smoking on pulmonary drug-metabolizing enzymes: a case-control study on lung cancer patients. Cancer Res 1988; 48:4695-700 9 Pryor WA, Prier DG, Church DF. Electron-spin resonance study of mainstream and sidestream cigarette smoke: nature of the free radicals in gas-phase smoke and in cigarette tar. Environ Health Perspect 1983; 47:345-55 10 Janoff A. Biochemical links between cigarette smoking and pulmonary emphysema. J Appl Physiol 1983; 55:285-93 11 Cerutti PA. Prooxidant states and tumor promotion. Science

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