Cancer Letters, 30 (1986) 289-297 Elsevier Scientific Publishers Ireland Ltd.

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INCREASED BINDING OF BENZO[a] PYRENE METABOLITES TO LYMPHOCYTES FROM PATIENTS WITH LUNG CANCER

LORRAE J. HAWKE and GEOFFREY C. FARRELL*

Department of Medicine, Westmead Hospital, Westmead, NSW 2145 (Australia)

(Received 6 November 1985) (Revised version received 17 January 1986) (Accepted 20 January 1986)

SUMMARY

This study was performed -to determine whether lymphocytes from individuals with lung cancer were more likely to bind benzo[a] pyrene (BP) metabolites in an in vitro test. The in vitro system consisted of peripheral blood lymphocytes incubated with increasing concentrations of [ 3H] benzo- [a] pyrene in the presence of @-naphthoflavone-induced rat liver microsomes and a NADPH-generating system. The radioactivity bound to a TCA-pre- cipitate of the lymphocytes was determined. The apparent affinity (Km) and maximal binding ( Vmax ) of this binding were calculated from double reciprocal plots of the data. Seven patients with primary lung cancer (squamous cell carcinoma and undifferentiated small cell carcinoma), none of whom had smoked for at least 2 months, were studied as were 10 lung cancer free smokers, and the results compared with 13 age- and sex-matched controls. In lymphocytes from patients with primary lung cancer, V,,, of radiolabel bound to TCA-precipitable material was almost double that of non-smoking controls (205 + 19.2 pmol - 2h - lo6 cells vs. 121 + 10.8 pmol - 2h l lo6 cells, mean f S.E.M., P< 0.01). Among individuals without lung cancer, smokers did not differ from non-smokers. In addition, there was no difference in Km of radiolabel binding to lymphocytes from patients in all 3 groups. It is concluded that binding of carcinogenic metabolites to cell components is increased in patients with lung cancer. Further studies are required to determine whether this increased binding is related to individual susceptibility of some smokers to lung cancer.

INTRODUCTION

Many epidemiological and other studies have implicated cigarette smoking as an important contributory factor in the development of lung cancer [ 7,271. The BP content of cigarette smoke is regarded as the most likely

*To whom all correspondence should be addressed.

0304-3335/861$03.50 0 1986 Elsevier Scientific Publishers Ireland Ltd. Published and Printed in Ireland

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component to produce this effect 119,281. BP is a polycyclic aromatic hydrocarbon (PAH) which, besides being found in relatively high concentra- tions in cigarette smoke, is also an ubiquitous environmental pollutant.

In order to exert its carcinogenic effect, BP must first be metabolized by the cytochrome P,,5,, -dependent enzyme aryl hydrocarbon hydroxylase (AHH) to reactive intermediates [ 91. A variety of intermediates are formed, which are either detoxified by epoxide hydrolase (EH) and conjugation with sulphate, glucuronic acid or glutathione, or are further oxidized to ultimate carcinogens such as the 7,8-dihydrodiol-9,10-epoxide [ 9,201. The binding of these reactive metabolites to DNA is thought to be a major step in carcino- genesis [ 11. It is thus evident that the balance between these activation and detoxification pathways of BP will determine the amount of ultimate carcinogen available for binding to DNA.

Not all smokers develop lung cancer. Family studies have provided evidence for an inherited susceptibility defect [ 51. It seems possible then, that a difference exists between individuals in their response to chronic BP ex- posure. We therefore have examined whether binding of BP metabolites to lymphocyte macromolecules differs between patients with lung cancer and controls. We also looked at whether cigarette smoking has a separate effect on such binding. Peripheral blood lymphocytes were chosen for this study as they are readily accessible, and known to possess AHH and the detoxification pathways [ 2,241.

MATERIALS AND METHODS

Subjects All subjects studied in this investigation were males over the age of 40

years. Group 1 comprised 13 controls who were either non-smokers or former smokers (from 13 months to 27 years before) without lung cancer. Two of these former smokers had had a colonic carcinoma resected. Group 2 com- prised 7 patients with primary lung cancer (squamous cell carcinoma and undifferentiated small cell carcinoma). All patients were former smokers (from 2 months to 20 years before). Two of these patients had cerebral metastases. Group 3 comprised 10 subjects currently smoking at least 20 cigarettes per day and with no cancer. The study protocol was approved by the Parramatta Hospitals Human Ethics Committee and all subjects gave informed consent.

Details concerning smoking habits, demographic factors, and therapeutic agents being taken by the subjects studied are provided in Table 1. The control group had a higher proportion of subjects taking therapeutic agents (including indomethacin, cimetidine, prednisolone and theophylline) at the time of the study which may interfere with BP metabolism [10,26], or who had occupational exposure to substances (including asbestos, coal dust, foundry fumes and diesel fumes) which may be synergistic with cigarette smoke in the development of lung cancer [ 15,17,21] . There were no signifi-

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cant differences in family history of lung or any other form of cancer, or in pack years of cigarette exposure (i.e. no. of packs of 20 cigarettes smoked per day X no. of years smoking) between the lung cancer group and controls. Only one subject in each of the control group and the lung cancer group had a family history of lung cancer.

In vitro binding assay Lymphocytes were isolated by standard procedures of layering diluted

venous blood over Ficoll-Paque (Pharmacia South Seas, Sydney, NSW). Lymphocytes were resuspended in medium (RPM1 1640 containing 2 mM L-glutamine and 20 mM HEPES) (Flow Laboratories Australasia, Sydney, NSW) at a concentration of 2 X 10” cells/ml.

Before preparation of microsomes, male Wistar rats (250 g) were injected i.p. with p-naphthoflavone (Sigma Chemical Co., St. Louis, MO) at 40 mg/kg body wt in maize oil daily for 3 days. Injections ceased 1 day before harvesting. Microsomes were prepared by standard procedures [8] except that aseptic techniques were used. Microsomal protein concentration was determined by the method of Lowry et al. [18] and adjusted to 10 mg/ml with medium.

The incubation mixture for the in vitro binding assay included the fol- lowing: [G-3H] benzo[a]pyrene (Amersham Australia Pty. Ltd., Sydney, NSW) diluted with unlabelled BP (Sigma Chemical Co., St. Louis, MO) to give a specific radioactivity between 150-180 dpm/pmol, and prepared in dime- thylsulphoxide (DMSO) to give 8 final concentrations ranging from 5 to 100 PM microsomal protein (0.5 mg), a NADPH-generating system comprising of NADP (0.4 mM), isocitrate (5.0 mM) and isocitrate dehydrogenase (units/ml) (Sigma Chemical Co., St. Louis, MO), and either 1 X lo6 lym- phocytes or an equivalent volume of medium. The final volume was 1.0 ml and this was incubated at 37°C for 2 h in a humidified chamber of 5% CO1 in air. Following this incubation, cells were centrifuged at 400 X g for 10 min at room temperature, washed in medium, then resuspended in 1 ml sterile, cold, distilled water and frozen at -20°C for l-3 days.

Cellular DNA and protein were precipitated as follows: the cells were thawed and 0.0125% BSA (Sigma Chemical Co., St. Louis, MO) added as carrier; TCA was then added to a final concentration of 5%. The precipitate was collected on glass fiber filters (Gelman Sciences Pty. Ltd. Inc., Sydney, NSW). Non-covalently bound BP metabolites were removed by washing the precipitate with 50 ml of hot (65°C) methanol.

The filters were then treated with NCS tissue solubilizer* (Amersham Australia Pty. Ltd., Sydney, NSW), and the radioactivity determined using Liquifluor scintillant (NEN, Boston, MA). Counts/min were converted to dpm by means of a quench curve.

Calculations and statistical analysis Disintigrations/min were converted to picomoles using the specific radio-

activity of the [3H] BP/BP preparation. Non-specific binding was determined

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by measurement of the picomoles bound in control tubes which did not contain lymphocytes. Specific binding was defined as total binding less non-specific binding. Specific binding during the 2-h incubation was plotted against the 8 concentrations of added BP. From a ‘double reciprocal plot of these data, the Vmax and K, were determined. All data used for the double reciprocal plots have a correlation coefficient r > 0.83 (P < 0.01).

Assays were performed in triplicate. V,,, and Km of the different groups were compared using the Wilcoxon-Mann-Whitney test, with P < 0.05 taken as significant. Data are expressed as mean + S.E.M.

RESULTS

It was apparent that the binding of radiolabel to TCA-precipitable material conformed to Michaelis-Menten kinetics (Fig. 1). The data were therefore analyzed using double reciprocal plots and V,,, and K, were thereby calculated for each group (Fig. 2)1 In patients with lung cancer, V,,, was found to be approximately twice that of the age- and sex-matched non- smoking controls (205 + 19.2 pmol - 2h - lo6 cells vs. 121 * 10.8 pmol * 2h * lo6 cells, mean f S.E.M., P < 0.01) However, there was no difference in Km between these 2 groups (Fig. 2). There was no difference in either V max or Km between smoking and non-smoking control groups (Fig. 2).

DISCUSSION

The results of this study demonstrate that lymphocytes from patients with primary lung cancer bind more BP metabolites than do those from age- and sex-matched controls. Cigarette smoking itself had no effect on this binding. In the present study there was no significant difference in pack years of cigarette exposure or other demographic features between the lung cancer group and controls. It would seem more likely therefore that some endogenous rather than exogenous factor(s) has resulted in these individuals being more susceptible to lung cancer than their matched controls.

Many factors affect individual susceptibility to cancer. Factors such as age [29], sex [22], hormonal status [lo], family history [5] and immune deficiency [6], have been shown to be relevant. Additionally, at the genetic and molecular level, differences in DNA repair, availability of carrier proteins and receptors for carcinogens [ll], differing isozyme composition of carcinogen metabolizing enzymes such as cytochrome Pd5,,, and imbalances between activating and detoxifying pathways due to insufficiencies of enzymes or cofactors [ 3,4,14,16,23,25] can determine individual susceptibility. In combination with causal agents such as dietary components [13], occupa- tional exposure [17], environmental pollutants [28], and smoking habits [7] the probability of an individual developing lung cancer can be altered.

The present data do not allow determination of whether the increased binding of BP metabolites to lymphocytes from lung cancer patients is related to their predisposition to develop lung cancer. An alternative ex- planation is that it is a secondary effect of the disease. A longitudinal study

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Fig. 1. Binding of benzo(a)pyrene metabolites to TCA-precipitable material conforms to MichaeligMenten kinetics. This binding curve represents the lung cancer group. The inset is the double reciprocal plot of these data, with a correlation coefficient of 0.99. Each point represents the mean of 7 values + S.E.M.

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of,individuals with high V,,, values for [3H] BP binding to lymphocytes would be of value to assess whether this high binding correlated prospectively with a high risk of developing lung cancer. Further studies might also assess BP binding to lymphocytes in patients with other types of cancer or examine this binding in patients with resected lung cancer.

In evaluating this in vitro assay, it must be kept in mind that (a) the lym- phocytes used may not behave the same way towards BP as the target cells in vivo, and (b) the BP metabolite profile generated in the presence of liver microsomes from rats treated with &naphthoflavone is probably different to that which the lung cells would be exposed to in vivo. It is also known that the blastogenic state of human lymphocytes affects their resting and inducible AHH levels [ 121, and it may be that the lymphocytes from the lung cancer patients were in a different blastogenic state compared to controls on account of their disease. Finally, the possible presence of yet undetected carcinoma in the smokers and ex-smokers of this study is also a complicating issue which should be kept in mind.

The aim of this study was to design a simple assay that could show if the binding of a carcinogen to cellular macromolecules varied between patients with lung cancer and controls. We in fact found increased binding of BP metabolites to macromolecules of lymphocytes from patients with lung

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cancer. At this stage in the development of this assay, the separation between the abnormal and normal ranges is incomplete such that on the basis of their V max values, approximately 50% of subjects would be falsely determined as ‘normal’.

Further attempts to reduce assay variability are now required together with prospective studies to determine whether the phenomenon we have observed is truly reflective of a susceptibility factor and can be used for identifying those among the smoking population at risk of developing lung cancer.

ACKNOWLEDGEMENTS

We are indebted to Drs. P. Despas, L. Engel, I. Gardiner, T. Robertson, P. Harvey and P. Gillespie for referring patients, to Debra Prendergast for technical assistance and to Dr. Michael Murray for his helpful advice. This project was supported by the Australian Tobacco’Research Foundation.

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