Lung Cancer and Past Occupational Exposure to Asbestos Role of p53 and K-ras Mutations Kirsti Husgafvel-Pursiainen, Antti Karjalainen, Annamaria Kannio, Sisko Anttila, Timo Partanen, Anneli Ojajärvi, and Harri Vainio Departments of Industrial Hygiene and Toxicology, Epidemiology and Biostatistics, and Occupational Medicine, Finnish Institute of Occupational Health, Helsinki, Finland; and International Agency for Research on Cancer, Lyon, France

Studies on somatic mutations in lung cancers associated with cigarette smoking and asbestos exposure are few. We investigated prevalence of mutations in the p53 and K-ras genes in lung tumors from smokers with and without asbestos exposure at work. For K-ras mutations, the study was an extension of an earlier analysis. Nearly all of the 105 consecutive patients examined were smokers and had non–small-cell carcinoma of the lung with squamous-cell carcinoma or adenocarcinoma histology. Exposure to asbestos was estimated by pulmonary fiber counts and occupational histories. A pulmonary burden of > 1 3 106 asbestos fibers per gram of lung tissue, indicating work-related exposure, was found in 32% of the patients for whom fiber-analysis data were available (33 of 102 patients, all men). The statistical analysis showed pulmonary fiber count as the only significant predictor of adenocarcinoma histology, in contrast to squamouscell carcinoma (smoking-adjusted odds ratio [OR] 3.0, 95% confidence interval [CI] 1.1 to 8.5). The frequency of p53 mutations was 39% (13 of 33) among the asbestos-exposed cases, as compared with 54% (29 of 54) among the nonexposed cases; the difference was not significant, however. In male ever-smokers, a long duration of smoking was associated with p53 mutation (OR 3.2, 95% CI 1.2 to 8.8). In adenocarcinoma, p53 mutations were less prevalent (10 of 30, 33%) as compared with squamous-cell carcinoma (28 of 46, 61%; P 5 0.02), whereas a strong and significant association was found between adenocarcinoma and K-ras mutation (OR 37, 95% CI 5.8 to 232, adjusted for smoking and asbestos exposure). Asbestos exposure alone was not significantly associated with increased occurrence of K-ras mutations. In conclusion, the results may primarily reflect the observed excess of adenocarcinoma in the asbestosexposed patients, and hence the decrease in p53 mutations and increase in K-ras mutations. HusgafvelPursiainen, K., A. Karjalainen, A. Kannio, S. Anttila, T. Partanen, A. Ojajärvi, and H. Vainio. 1999. Lung cancer and past occupational exposure to asbestos: role of p53 and K-ras mutations. Am. J. Respir. Cell Mol. Biol. 20:667–674.

An association between past exposure to asbestos and lung cancer has been epidemiologically established since the 1970s, and asbestos is considered a human carcinogen (1, 2). The highest risks have been observed in occupations with continuing heavy exposure to friable asbestos materials, insulation work being one example. Considerable exposure to asbestos has also occurred in construction and (Received in original form April 29, 1998 and in revised form August 6, 1998) Address correspondence to: Dr. Kirsti Husgafvel-Pursiainen, Laboratory of Molecular and Cellular Toxicology, Dept. of Industrial Hygiene and Toxicology, Finnish Institute of Occupational Health, Topeliuksenkatu 41 aA, FIN-00250 Helsinki, Finland. E-mail: Kirsti.Husgafvel-Pursiainen@ occuphealth.fi Abbreviations: confidence interval, CI; fibers per gram, f/g; odds ratio, OR; scanning electron microscopy, SEM. Am. J. Respir. Cell Mol. Biol. Vol. 20, pp. 667–674, 1999 Internet address: www.atsjournals.org

shipyard occupations (3). Although many countries have taken actions to restrict or ban the production and use of asbestos, concern about the health risks of asbestos exposure continues because of high exposures in the past and uncontrolled asbestos removal, as well as continued production and consumption in other parts of the world (4). In 1990, the world production of asbestos was still more than 1.5 million tons (5), and the use of asbestos causes a widespread hazard in developing countries (6). Chemical carcinogenesis is seen as a multifactorial, multistage process that takes place over a relatively long period of time. Several factors are known or suspected to affect the cancer risk associated with asbestos exposure. Co-exposure to tobacco smoke results in multiplication of the risk (2, 7). The type and dimensions, as well as biologic persistence and durability, of the fibers are important in fiber carcinogenesis (8). Typically, a latency period of at least 15 to 20 yr between the exposure and clinical mani-

668

AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL. 20 1999

festation of the disease is observed. The roles of pulmonary fibrosis (asbestosis) and asbestos fibers per se have also been discussed (3, 9). Finally, the efficacy of asbestos exposure as a complete human carcinogen independent of tobacco smoke has not been entirely settled. Most lung cancers of asbestos-exposed individuals occur in smokers and ex-smokers, and even in large cohort studies the risk estimates for lifetime nonsmokers are based on relatively few lung cancer cases (10). Despite many decades of research on asbestos-related pulmonary carcinogenesis, the molecular mechanisms involved have remained largely unknown. Experimental studies have shown that asbestos fibers are genotoxic; they are able to induce DNA damage, chromosome aberrations, mitotic disturbances, and gene mutations (for review, see [11]). In addition, asbestos fibers can stimulate cell proliferation and chronic inflammation (8, 12), as well as induce gene expression (13, 14) and malignant transformation of human cells (15). Mutations in the p53 tumor suppressor gene are the most common genetic alterations observed in human cancer, and they are particularly frequent in lung cancer (16). There are several well-known examples of human cancers in which an association between carcinogenic exposure and p53 mutations has been observed, thus supporting involvement of the p53 mutational pathway in carcinogenesis associated with external exposures (16–18). This background prompted us to examine the occurrence of p53 and K-ras mutations in lung tumors from smokers who had been occupationally exposed to asbestos for several years in the past. The mutation data were then analyzed with regard to tumor histology, tobacco smoking, and occupational asbestos exposure. We used tumor cell type, the amount and duration of tobacco smoking, time since quitting smoking, asbestos exposure estimated on the basis of occupational histories, and pulmonary asbestos fiber counts as possible determinants.

Materials and Methods Lung Cancer Patients The study population consisted of 105 consecutive lung cancer patients who underwent surgical lobectomy or pulmectomy at the Department of Thoracic and Cardiovascular Surgery at the Helsinki University Hospital (Helsinki, Finland) between August 1988 and October 1992. Most of the patients were smokers. Detailed smoking data were available for 101 patients: 62 (61%) were current smokers; 35 patients (35%) had stopped smoking at least one year before surgery, and 21 of them for five or more years before the operation; four patients (4%) had never smoked. Smoking details were not available for four patients. Eighty-nine men, mean 6 SD age of 63 6 8 yr (range, 35 to 80), and 16 women, mean age of 65 6 7 yr (range, 43 to 75) were included. Histopathology showed 53 squamouscell carcinomas (47 men and 6 women), 39 adenocarcinomas (30 men and 9 women), five large-cell carcinomas (all men), seven small-cell carcinoma (six men and one woman), and one male adenosquamous carcinoma case. About 65% of the tumors were of stage I–II, and 30% of stage IIIa. Because surgical patients were selected, the proportion of

small-cell lung cancers was lower than among lung cancer patients in general. The tumor specimens were also histologically investigated for pulmonary fibrosis (asbestosis) (19). The study was approved by the ethics committees of Helsinki University Central Hospital and the Finnish Institute of Occupational Health (Helsinki, Finland). A physician experienced in occupational medicine (A. Karjalainen) interviewed all patients in person at the hospital. Each patient gave informed consent before the interview. Occupational Exposure and Smoking Complete work history, including past occupational, domestic, and environmental exposure to asbestos, was obtained for each patient at the interview. On the basis of these data, the probability of occupational exposure to asbestos was evaluated by two occupational hygienists by consensus. This was done without any knowledge of the asbestos counts from the tissue samples. An exposure period of 1 mo was regarded as a minimum. Each patient was classified into one of the four exposure categories (definite, probable, possible, unlikely), as described (20). In addition, the frequency and duration of the tasks with at least probable exposure to asbestos were also considered in the classification. The personal interview also included questions on the patient’s smoking history: Age at starting and quitting smoking, nonsmoking periods longer than 6 mo, and the daily consumption of cigarettes/cigars/pipes were recorded. Only three patients had used tobacco products other than cigarettes (pipe). Lung Tissue Samples and Fiber Analysis The tissue pieces for electron microscopic fiber analysis were taken from the peripheral part of the lung and did not include pleural or tumor tissue. In case of bilobectomy or pulmectomy, the sample was taken from the lobe that appeared to be closest to normal. Organic tissue was removed from a tissue piece of about 100 mg wet weight by low-temperature ashing. Fibers were detected with a JEOL 100 CX-ASID4D electron microscope (JEOL Ltd., Tokyo, Japan) in scanning electron microscopy (SEM) mode at 5,000-fold magnification. A length-to-width ratio greater than 3 and roughly parallel sides were used as criteria for fiber. Fibers longer than 1 mm could be detected. At minimum, 200 viewing fields were evaluated to find at least four to 30 fibers per sample, depending on the density. An analytical sensitivity (one fiber per sample) of about 0.1 3 106 fibers per gram (f/g) dry tissue could be reached. An energy-dispersive X-ray microanalyzer (Tracor TN 5500; Noran Instruments, Middleton, WI) was used to determine the fiber type by comparing peak ratios with standard spectra. The total pulmonary concentration of asbestos fibers was used as an indicator of past asbestos exposure. Chrysotile fibers are poorly detected with SEM and, consequently, the results represent the concentration of amphibole fibers. On the basis of previous studies, a concentration exceeding 1.0 3 106 f/g was considered highly indicative of past occupational exposure to asbestos (21). Tissue Samples and Analysis of p53 and K-ras Genes Representative samples of fresh tumor tissue were provided by the pathologist and frozen at 2808C for future

Husgafvel-Pursiainen, Karjalainen, Kannio, et al.: p53 Mutations and Asbestos Exposure

mutation analysis. DNA was extracted from the tissue sample by phenol–chloroform extraction as described elsewhere (22). All lung tumor samples were screened for p53 alterations using denaturant gradient gel electrophoresis (DGGE). Polymerase chain reaction (PCR)–DGGE of exons 5–9 as well as the procedures and sequencing primers for direct sequencing were the same as given previously (22–24). At minimum, two to three separate PCR products from each sample were sequenced to confirm the result. Activating point mutations in the K-ras gene exons 1 and 2 (codons 12, 13, 61) were examined from the same tissue samples as p53 gene mutations; the methods and details have been given elsewhere (25, 26). Altogether, 97 lung tumors were studied for K-ras mutations; the present study is an extension of a previously published analysis (26). Statistical Analysis Bivariate and multivariate logistic regression models were applied in quantifying the associations between end-point parameters (lung cancer cell type, and p53 or K-ras mutations) and indicators of previous exposure to asbestos and to tobacco smoke. Past exposure to asbestos was analyzed using pulmonary fiber burden ( , 1.0 3 106 f/g versus > 1.0 3 106 f/g) as the criterion for occupational exposure. Because none of the female patients had been exposed to asbestos at work, only men were included in the analyses. All P values given are one-sided.

Results Asbestos Exposure of the Patients Demographic and background data for cigarette smoking and asbestos exposure of the patients are presented in Table 1. Thirty-three patients (32%, all men) out of the 102 for whom fiber-analysis data were available showed a pulmonary deposition of 1 million or more amphibole asbestos fibers per gram of dry lung tissue (range 1.0 to 145 3 106 f/g); this is considered highly indicative of occupational exposure to asbestos. Antophyllite was the main type of

669

asbestos fiber detected. Job history data on these patients indicated work-related exposure in the past: 30 of the 33 patients with a history of asbestos exposure were classified as definitely, probably, or possibly exposed. Of these, seven patients were diagnosed with asbestosis. For 69 patients, the pulmonary fiber concentration was below 1 million (range , 0.3 to 0.95 3 106 f/g; Table 1). Tumor Histology Of the cases not exposed to asbestos, 59% were squamous-cell carcinomas and 26% adenocarcinomas. For the exposed cases, the corresponding prevalences were 42% and 48%, respectively; that is, the frequency of adenocarcinoma was almost twofold (Figure 1). In the statistical analysis adjusted for smoking parameters, 1 million or more asbestos fibers per gram of dry lung tissue emerged as the strongest and only significant predictor of adenocarcinoma, as contrasted to squamous-cell carcinoma (smoking-adjusted odds ratio [OR] 3.0, 95% confidence interval [CI] 1.1 to 8.5; Table 2). Smoking history (i.e., lifetime daily amount of cigarettes, duration, starting age, or years since quitting) was not significantly associated with either histology. p53 Mutations In all, 51% (54 of 105) of the patients exhibited a somatic p53 mutation. The mutation analysis showed that 39% (13 of 33) of the occupationally exposed male cases had a p53 mutation, as compared with 54% (29 of 54) of the nonexposed male cases. The prevalence of mutations tended to decrease with the increasing amounts of fibers in the lungs, being only 20% (2 of 10) among the cases with the highest pulmonary asbestos-fiber content (> 5 3 106 f/g) (Figure 1). The difference remained, however, statistically nonsignificant (Table 3). Of the exposed cases with histologic as-

TABLE 1

Demographic and exposure data on lung cancer patients by pulmonary asbestos fiber concentration Pulmonary Concentration of Asbestos Fibers > 1.0 3 106 , 1.0 3 106 (f/g) (f/g)

Sex (M/F) Age at diagnosis Smoking (mean 6 SD) Cigarettes smoked/d Pack-years Yr since quitting Age at starting Pulmonary fiber content Mean 6 SD Median

33/0 64 6 7

54/15* 63 6 9

25 6 12 44 6 24 6.8 6 12 18 6 4.3

20 6 9 41 6 22 2.6 6 6.3 18 6 6.0

11 6 28 2.2

0.3 6 0.2† 0.3

* For one female patient, no data available for asbestos exposure. † Fiber concentration below detection limit (0.3 3 106 f/g) taken into account as 0.5 3 0.3 3 106 f/g.

Figure 1. Prevalence of adenocarcinoma histology (%), p53 mutations, and K-ras mutations in male lung cancer patients with (pulmonary asbestos fiber concentrations 1 to , 5 3 106 and > 5 3 106 f/g dry weight) and without (, 1 3 106 f/g) occupational exposure to asbestos.

670

AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL. 20 1999

TABLE 2

Uni- and multivariate prediction of histologic type (adenocarcinoma versus squamous-cell carcinoma) with indicators of occupational asbestos exposure and tobacco smoking Univariate Predictor

Asbestos exposure (> 1 million f/g dry wt of lung) Mean lifetime daily smoking (. 20 cigarettes/d) Duration of smoking (. 43 yr) Time from cessation of smoking to clinical appearance (> 5 yr) Age at onset of smoking (, 18 yr)

Multivariate

NEC

OR

95% CI

OR

95% CI

15

2.99

1.10–8.09

3.03

1.09–8.47

7 10

0.83 0.85

0.28–2.45 0.32–2.28

0.61 0.93

0.18–2.03 0.32–2.69

8 14

1.43 1.29

0.48–4.42 0.50–3.38

1.27 1.29

0.40–4.04 0.45–3.66

Includes ever-smoker men, adenocarcinoma, and squamous-cell carcinoma histologies only. All predictors are included in the multivariate models. Excludes subjects with missing data in any variable in the analysis. Predictor, exposed category given in parentheses. NEC, number of exposed cases.

bestosis, two (2 of 7, 29%) had a p53 mutation. Interestingly, mutations in p53 gene were significantly less prevalent in adenocarcinoma (10 of 30, 33%), the cell type associated here with asbestos exposure, as compared with squamous-cell carcinoma (28 of 46, 61%; P 5 0.02). The locations and types of mutations detected in the asbestos-exposed cases are presented in Table 4. The mutations seen were primarily missense and nonsense mutations; one deletion causing a frameshift had also occurred. Exon 5 was the most frequently mutated region (5 of 13, 38%), with two identical G-to-C transversions at codon 181. In all, among the exposed, six base substitutions had occurred at G:C base pairs (6 of 12, 50%), including two G:C-to-T:A transversions (17%) and one G:C-to-A:T transition at a non-CpG site. In addition, three (3 of 12; 25%) A:T-to-G:C transitions were detected; altogether five base substitutions had occurred at A:T pairs (5 of 12, 42%) (Table 4). The nonexposed smokers carried 39 mutations, 32 of which were identified by sequencing. This group showed 10 (31%) G-to-T transversions on the nontranscribed strand, a mutation characteristic to tobacco-smoke exposure. In smokers, the most frequently mutated codons

were as follows: codons 155 and 173 had mutated twice, codon 245 three times, and codon 273 twice. Of these, codons 245 and 273 are included in the hot-spot mutational sites of lung cancer. In addition, hot-spot codons 157, 158, 179, 248, and 282 were all mutated once. Thus, the lung cancer–associated hot-spot codons harbored 31% (10 of 32) of the mutations observed in nonexposed smokers as compared with one (8%) in the asbestos-exposed cases. One feature of the study population was that one-third of the asbestos-exposed cases were long-term ex-smokers. Among male cases, the highest rate of mutations (53%) was observed among current smokers without occupational exposure, and the rate was somewhat lower among the men who had quit smoking > 5 yr before the operation (Table 5). The statistical analysis revealed long duration of smoking as a statistically significant predictor of p53 mutation (Table 3). K-ras Mutations The frequency of patients with K-ras mutations was 21% (20 of 97). In men, adenocarcinoma histology showed strong and significant association with K-ras mutation (OR 37, 95% CI 5.8 to 232; Table 6). Mutation frequency

TABLE 3

Uni- and multivariate prediction of p53 mutations with histologic type and indicators of occupational asbestos exposure and tobacco smoking Univariate Predictor

Histologic type (adenocarcinoma) Asbestos exposure (> 1 million f/g dry wt of lung) Mean lifetime daily smoking (. 20 cigarettes/d) Duration of smoking (. 43 yr) Time from cessation of smoking to clinical appearance (> 5 yr) Age at onset of smoking (, 18 yr) See footnotes to Table 2.

Multivariate

NEC

OR

95% CI

OR

95% CI

10

0.41

0.15–1.09

0.42

0.14–1.28

11

0.47

0.18–1.23

0.69

0.23–2.06

7 19

0.41 3.23

0.15–1.10 1.19–8.79

0.58 2.87

0.17–2.00 0.98–8.40

7 13

0.53 0.38

0.18–1.57 0.14–0.98

0.82 0.45

0.24–2.78 0.16–1.31

Husgafvel-Pursiainen, Karjalainen, Kannio, et al.: p53 Mutations and Asbestos Exposure

671

TABLE 4

Types of p53 mutations in lung cancer patients with occupational asbestos exposure p53 Mutations Case No.

185 129 121 11 28 175 165 191 143 12 102 30 40

Age

Pulmonary Fiber Content*

Smoking (py/ex)†

Exon

Codon

Base Change

Amino Acid Change

65 71 58 69 63 64 65 63 73 60 73 64 58

5.9 1.4 2.1 2.0 2.9 1.9 1.0 145.0 3.5 2.2 1.6 1.3 2.1

32/0 35/0 16/8 58/2 ND 70/18 45/0 22/20 27/0 50/0 45/48 41/3 20/0

5 5 5 5 5 6 6 7 8 8 8 8 9

155 166 171 181 181 214 220 246 270 273 286 ND§ 317

ACC . CCC TCA . TGA GAG . TAG CGC . CCC CGC . CCC CAT . CGT TAT . TGT ATG . GTG TTT . delT CGT . CAT‡ GAA . TAA

Thr . Pro Ser . Stop Glu . Stop Arg . Pro Arg . Pro His . Arg Tyr . Cys Met . Val Frame shift Arg . His Glu . Stop

CAG . CTG

Gln . Leu

* 3 10 f/g dry weight of lung tissue. † Cigarette smoke exposure in pack-yr (py)/time (yr) since cessation of smoking (ex). ‡ G:C . A:T mutation at CpG site. § No sequence data available. 6

was 33% (10 of 30) in cases with occupational asbestos exposure, and 17% (9 of 54) in the nonexposed cases (P 5 0.07). The frequency seen in the exposed cases with histologic asbestosis was 29% (2 of 7). The distribution of K-ras mutations according to pulmonary asbestos-fiber content is shown in Figure 1. The univariate analysis suggested an increased probability of K-ras mutations in the occupationally exposed cases, but the OR came close to unity in the multivariate analysis (Table 6). The K-ras mutation frequency roughly followed the daily amount of cigarettes smoked; this was seen particularly in men. The prevalence of K-ras–positive male cases was 13% (2 of 16) in smokers who smoked , 20 cigarettes/d, 21% (6 of 29) in those smoking 20 to 29 cigarettes/d, and 36% (5 of 14) in those who consumed > 30 cigarettes/d. The differences were suggestive, but not statistically significant (P for trend, 0.07).

Discussion In this work, 105 lung cancer patients (mainly men with non–small-cell lung cancer of stage I–IIIa) who were con-

TABLE 5

Prevalence of p53 mutations in male lung cancer patients by occupational asbestos exposure and smoking status Exposure

Nonexposed Current smokers Long-term ex-smokers Asbestos-exposed Current smokers Long-term ex-smokers

Cases Analyzed (n)

Cases with p53 Mutation (n)

(%)

34 11

18 5

53 45

16 10

6 4

38 40

Long-term ex-smoker, > 5 yr since cessation.

secutively admitted to hospital for a curative operation were examined for the presence of mutations in the p53 tumor suppressor gene and K-ras oncogene. Of the cases studied, 33 had been exposed to asbestos at work, based on pulmonary asbestos-fiber counts determined by SEM and in-person interviews. The results indicated that lung tumors with p53 mutation were not more common among the cases with occupational asbestos exposure (39%) than in the nonexposed cases (54%). The group with the heaviest occupational exposure appeared to have a lower prevalence of p53 mutations (20%) than the patients with lower fiber counts (48%). The results suggested a negative correlation between pulmonary fiber burden and p53 mutation frequency, but the OR remained nonsignificant in the multivariate analysis. In previous studies, an association between p53 protein overexpression or mutations and asbestos exposure has been suggested (27–30). The published studies deviate from the present one in some respects. First, in contrast to the other studies, we used the concentration of asbestos fibers measured in the lung tissue for documentation of the occupational exposure. In addition, work history was used for probability and timing of the exposure. Second, the earlier studies examined relatively few exposed cases, whereas we studied cases from an industrial area with onethird of our lung cancer population occupationally exposed to asbestos. In agreement with the present findings, preliminary results from a study that used asbestos body measurements and questionnaire-based exposure classification as the exposure indicators did not find accumulation of p53 protein among the exposed lung cancer cases (31). Previously, lung cancers from subjects with asbestosinduced fibrosis, indicating heavy asbestos exposure, did not show increased p53 mutation frequency or protein expression (24). Experimental animal studies have also shown conflicting results. Expression of p53 was induced in the lungs of chrysotile asbestos–treated rats but not in

672

AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL. 20 1999

TABLE 6

Uni- and multivariate prediction of K-ras mutations with histologic type and indicators of occupational asbestos exposure and tobacco smoking Univariate Predictor

Histologic type (adenocarcinoma) Asbestos exposure (> 1 million f/g dry wt of lung) Mean lifetime daily smoking (. 20 cigarettes/d) Duration of smoking (. 43 yr) Time from cessation of smoking to clinical appearance (> 5 yr) Age at onset of smoking (, 18 yr)

Multivariate

NEC

OR

95% CI

OR

95% CI

15

24.4

4.87–122

36.6

5.79–232

9

2.25

0.74–6.87

1.02

0.22–4.71

7 5

2.05 0.60

0.65–6.48 0.18–1.94

3.93 0.53

0.58–26.7 0.11–2.65

4 10

0.90 1.74

0.25–3.25 0.57–5.29

0.49 1.13

0.09–2.74 0.23–5.66

See footnotes to Table 2.

the control rats exposed to iron beads (32). Another study reported lack of p53 mutations in crocidolite-induced tumors, whereas treatment with benzo(a)pyrene resulted in tumors with p53 mutations (33). The types of base substitutions observed here in the exposed versus nonexposed cases had different features. In the exposed group, half of the mutations resided at G:C pairs, but only two (17%) were G:C-to-T:A transversions on the nontranscribed strand. In contrast, 30% of the mutations observed in the nonexposed cases were G-to-T transversions, a frequency comparable with findings from other studies and the p53 mutation database (16, 34). G:Cto-T:A is a base substitution typically seen in lung cancer, and is indicated as a hallmark of exposure to tobacco smoke (16) and to benzo(a)pyrene in particular (35). Another source for base modifications at G:C pairs in both groups may be oxygen free radicals. Exposure to tobacco smoke and asbestos fibers both result in generation of oxidative damage (36, 37), especially in formation of 8-OHdeoxyguanosine (38, 39), which in turn causes primarily G-to-T transversions, but other substitutions are detected as well (40, 41). Here the small frequency of G-to-T transversions detected in the asbestos-exposed cases did not speak in favor of enhancement of this mechanism in association with co-exposure. Previously, a predominance of G-to-T transversions in asbestos-exposed lung cancers has been reported (28, 29), but not in all studies (30). G:C-toA:T transitions were rare (1 of 12, 8%) in the present exposed cases, but represented the second most frequent type of mutation in the nonexposed smokers (totally, 8 of 33, 24%; at non-CpG, 6 of 8). This is in keeping with the hypothesis and previous data that G:C-to-A:T transitions in cancers associated with external exposure characteristically occur at non-CpG sites (16). Finally, because asbestos fibers induce DNA strand breaks (11), deletions may be expected as typical mutation. However, only one deletion was seen in the exposed lung cancers. Reasons for the observed lower occurrence of p53 mutations among the asbestos-exposed male lung cancers compared with the nonexposed smoking ones may be multiple. At cellular level, not only tobacco smoke constituents but also asbestos fibers induce DNA damage (11) and

apoptosis (42, 43), both important factors in carcinogenesis and tumor formation. Because p53 controls cell growth and division by being a sensor of DNA damage and a mediator of apoptosis (44), one would expect that cellular pathways leading to loss of p53 function due to mutations would be prominent in smoking- and asbestos-related carcinogenesis. Asbestos fibers can nevertheless affect regulation of transcription and gene expression via many other cellular mediators (8, 13, 14, 45–47), an observation that may suggest a less significant role for selection for somatic p53 mutations. In agreement with this, p53 mutations have infrequently been seen in human malignant mesothelioma, a pulmonary malignancy related to asbestos exposure (48, 49). In adenocarcinoma, a strong and significant association between this cell type and K-ras mutation was observed, whereas p53 mutations were less frequent in comparison with squamous-cell carcinoma. The relationship between adenocarcinoma and K-ras mutations has been reported previously (50, 51), with the suggestion that mutations may occur in adenocarcinoma frequently enough to be used as a molecular marker in clinical work (52). The frequency of K-ras mutations showed an increasing trend with increasing daily smoking, and mutations were more frequent in the exposed than in the nonexposed cases, as was the case in a subset of the present study population (26). After adjustment for smoking and histology, the association between K-ras mutations and asbestos exposure remained, however, nonsignificant. With regard to lung cancer histology and exposure to asbestos, the study showed a significant association between high pulmonary asbestos-fiber content and adenocarcinoma histology. A preponderance of adenocarcinomas has been observed among asbestos-exposed lung cancer patients in some but not all previous studies (53– 57). Recently, a large, cancer registry–based follow-up of 1,300 men with asbestosis and 4,700 men with benign asbestos-related pleural disease was carried out (58). The results indicated a clearly increased risk for all main histologic types of lung cancer in male patients with asbestosis. The risk estimates were, however, somewhat higher for adenocarcinoma and small-cell carcinoma than for squa-

Husgafvel-Pursiainen, Karjalainen, Kannio, et al.: p53 Mutations and Asbestos Exposure

mous-cell carcinoma in both groups (58). Selection of patients and diagnostic procedure (surgery, bronchoscopy, autopsy), as well as variation in the mean intensity of asbestos exposure, may influence the results obtained from the various studies. For example, none of the female patients in our study had been occupationally exposed to asbestos. Our analyses were therefore restricted to men only. Also, because the study comprised surgical cases of lung cancer, only two histologic types of non–small-cell lung cancer (adenocarcinoma and squamous-cell carcinoma) were taken into account. Consequently, no direct conclusions can be drawn from the present results on female lung cancer or small-cell lung cancer. In conclusion, we propose that the observed differences in the frequencies of p53 and K-ras mutations between the asbestos-exposed and nonexposed lung cancer cases may, at least partially, reflect the relative increase of adenocarcinoma cell type among the exposed cases. Acknowledgments: The authors thank Ms. Tuula Suitiala for excellent technical assistance, and Ms. Terttu Kaustia, M.A., for language revision. Partial financial support from the Ministry of the Environment, Finland (Dnr. 17/742/94), and the EU/Environment programme, DG XII of the Commission of the European Communities (project EV5V-CT94-0555) is gratefully acknowledged.

References 1. Doll, R., and R. Peto. 1981. The Causes of Cancer: Quantitative Estimates of Avoidable Risks of Cancer in the United States Today. Oxford University Press, Oxford, New York. 2. IARC. 1987. Monographs on the Evaluation of Carcinogenic Risks to Humans. Overall Evaluations of Carcinogenicity: An Updating of IARC Monograph Vol. 1 to 42, Suppl. 7. International Agency for Research on Cancer, Lyon, France. 106–116. 3. Wilkinson, P., D. M. Hansell, J. Janssens, M. Rubens, R. M. Rudd, A. Newman-Taylor, and C. McDonald. 1995. Is lung cancer associated with asbestos exposure when there are no small opacities on the chest radiograph? Lancet 345:1074–1078. 4. Karjalainen, A. 1997. Asbestos: a continuing concern. Scand. J. Work Environ. Health 23:81–82. 5. Anonymous. 1992. Asbestos production: the chrysotile crisis. Industrial Minerals May:41–53. 6. Levy, B. S., and A. Seplow. 1992. Asbestos-related hazards in developing countries. Environ. Res. 59:167–174. 7. Hammond, E. C., I. J. Selikoff, and H. Seidman. 1979. Asbestos exposure, cigarette smoking and death rates. Ann. NY Acad. Sci. 330:390–473. 8. IARC. 1996. In Mechanisms of Fibre Carcinogenesis. A. B. Kane, P. Boffetta, R. Saracci, and J. D. Wilbourn, editors. International Agency for Research on Cancer, Lyon. IARC Scientific Publications No. 140. 9. Hughes, J. M., and H. Weill. 1991. Asbestosis as a precursor of asbestosrelated lung cancer: results of a prospective mortality study. Br. J. Ind. Med. 48:229–233. 10. Vainio, H., and P. Boffetta. 1994. Mechanisms of the combined effect of asbestos and smoking in the etiology of lung cancer. Scand. J. Work Environ. Health 20:235–242. 11. Jaurand, M.-C. 1997. Mechanisms of fiber-induced genotoxicity. Environ. Health Perspect. 105:1073–1084. 12. Walker, C., J. Everitt, and J. C. Barrett. 1992. Possible cellular and molecular mechanisms for asbestos carcinogenicity. Am. J. Ind. Med. 21:253–273. 13. Heintz, N. H., Y. M. W. Janssen, and B. T. Mossman. 1993. Persistent induction of c-fos and c-jun expression by asbestos. Proc. Natl. Acad. Sci. USA 90:3299–3303. 14. Timblin, C. R., Y. W. M. Janssen, and B. T. Mossman. 1995. Transcriptional activation of the proto-oncogene c-jun by asbestos and H2O2 is directly related to increased proliferation and transformation of tracheal epithelial cells. Cancer Res. 55:2723–2726. 15. Hei, T. K., L. J. Wu, and C. Q. Piao. 1997. Malignant transformation of immortalized human bronchial epithelial cells by asbestos fibers. Environ. Health Perspect. 105:1085–1088. 16. Greenblatt, M. S., W. P. Bennett, M. Hollstein, and C. C. Harris. 1994. Mutations in the p53 tumor suppressor gene: clues to cancer etiology and molecular pathogenesis. Cancer Res. 54:4855–4878. 17. Puisieux, A., S. Lim, J. Groopman, and M. Ozturk. 1991. Selective targeting of p53 gene mutational hotspots in human cancers by etiologically defined carcinogens. Cancer Res. 51:6185–6189.

673

18. Biggs, P. J., W. Warren, S. Venitt, and M. R. Stratton. 1993. Does a genotoxic carcinogen contribute to human breast cancer? The value of mutational spectra in unravelling the aetiology of cancer. Mutagenesis 8:275– 283. 19. Craighead, J. E., J. L. Abraham, A. Churg, F. H. Y. Green, J. Kleinerman, P. C. Pratt, T. A. Seemayer, V. Vallyathan, and H. Weill. 1982. The pathology of asbestos-associated diseases of the lungs and pleural cavities: diagnostic criteria and proposed grading schema. Arch. Pathol. Lab. Med. 106: 544–596. 20. Karjalainen, A., S. Anttila, L. Heikkilä, P. Karhunen, and H. Vainio. 1993. Asbestos exposure among Finnish lung cancer patients: occupational history and fiber concentration in lung tissue. Am. J. Ind. Med. 23:461–471. 21. Karjalainen, A., E. Vanhala, P. Karhunen, K. Lalu, A. Penttilä, and A. Tossavainen. 1994. Asbestos exposure and pulmonary fiber concentration of 300 Finnish urban men. Scand. J. Work Environ. Health 20:34–41. 22. Ridanpää, M., A. Karjalainen, S. Anttila, H. Vainio, and K. Husgafvel-Pursiainen. 1994. Genetic alterations in p53 and K-ras in lung cancer in relation to histopathology of the tumor and smoking history of the patient. Int. J. Oncol. 5:1109–1117. 23. Kannio, A., M. Ridanpää, H. Koskinen, T. Partanen, S. Anttila, Y. Collan, E. Hietanen, H. Vainio, and K. Husgafvel-Pursiainen. 1996. A molecular and epidemiological study on bladder cancer: p53 mutations, tobacco smoking, and occupational exposure to asbestos. Cancer Epidemiol. Biomarkers Prev. 5:33–39. 24. Husgafvel-Pursiainen, K., A.-M. Kannio, P. Oksa, T. Suitiala, H. Koskinen, R. Partanen, K. Hemminki, S. Smith, R. Rosenstock-Leiby, and P. W. Brandt-Rauf. 1997. Mutations, tissue accumulations, and serum levels of p53 in patients with occupational cancers from asbestos and silica exposure. Environ. Mol. Mutagen. 30:224–230. 25. Ridanpää, M., and K. Husgafvel-Pursiainen. 1993. Denaturing gradient gel electrophoresis (DGGE) assay for K-ras and N-ras genes: detection of K-ras point mutations in human lung tumour DNA. Hum. Mol. Genet. 2:639–644. 26. Husgafvel-Pursiainen, K., P. Hackman, M. Ridanpää, S. Anttila, A. Karjalainen, T. Partanen, O. Taikina-aho, L. Heikkilä, and H. Vainio. 1993. K-ras mutations in human adenocarcinoma of the lung: association with smoking and occupational exposure to asbestos. Int. J. Cancer 53:250–256. 27. Nuorva, K., R. Mäkitaro, E. Huhti, D. Kamel, K. Vähäkangas, R. Bloigu, Y. Soini, and P. Pääkkö. 1994. p53 protein accumulation in lung carcinomas of patients exposed to asbestos and tobacco smoke. Am. J. Respir. Crit. Care Med. 150:528–533. 28. Guinee, D. G., W. D. Travis, G. E. Trivers, V. M. G. De Benedetti, H. Cawley, J. A. Welsh, W. P. Bennett, J. Jett, T. V. Colby, H. Tazelaar, S. L. Abbondanzo, P. Pairolero, V. Trastek, N. E. Caporaso, L. A. Liotta, and C. C. Harris. 1995. Gender comparisons in human lung cancer: analysis of p53 mutations, anti-p53 serum antibodies and C-erbB-2 expression. Carcinogenesis 16:993–1002. 29. Wang, X., D. C. Christiani, J. K. Wiencke, M. Fischbein, X. Xu, T. J. Cheng, E. Mark, J. C. Wain, and K. T. Kelsey. 1995. Mutations in the p53 gene in lung cancer are associated with cigarette smoking and asbestos exposure. Cancer Epidemiol. Biomarkers Prev. 4:543–548. 30. Harty, L. C., D. G. Guinee, Jr., W. D. Travis, W. P. Bennett, J. Jett, T. V. Colby, H. Tazelaar, V. Trastek, P. Pairolero, L. A. Liotta, C. C. Harris, and N. E. Caporaso. 1996. P53 mutations and occupational exposures in a surgical series of lung cancers. Cancer Epidemiol. Biomarkers Prev. 5:997– 1003. 31. Pairon, J. C., M. A. Billon-Galland, Y. Iwatsubo, I. Abd-Al-Samad, P. Brochard, C. Danel, F. Galateau-Salle, B. Housset, M. Letourneux, R. Lubin, I. Monnet, J. F. Regnard, M. Riquet, and T. Soussi. 1997. P53 expression and asbestos exposure in lung cancer. Eur. Respir. J. 10:S111. (Abstr.) 32. Mishra, A., J.-Y. Liu, A. R. Brody, and G. F. Morris. 1997. Inhaled asbestos fibers induce p53 expression in the rat lung. Am. J. Respir. Cell Mol. Biol. 16:479–485. 33. Unfried, K., N. Kociok, M. Roller, J. Friemann, F. Pott, and W. Dehnen. 1997. P53 mutations in tumours induced by intraperitoneal injection of crocidolite asbestos and benzo[a]pyrene in rats. Exp. Toxic. Pathol. 49: 181–187. 34. Hainaut, P., T. Hernandez, A. Robinson, P. Rodriguez-Tome, T. Flores, M. Hollstein, C. C. Harris, and R. Montesano. 1998. IARC database of p53 gene mutations in human tumors and cell lines: updated compilation, revised formats and new visualisation tools. Nucleic Acids Res. 26:205–213. 35. Denissenko, M. F., A. Pao, M.-S. Tang, and G. P. Pfeifer. 1996. Preferential formation of benzo[a]pyrene adducts at lung cancer mutational hotspots in p53. Science 274:430–432. 36. Mossman, B. T., J. Bignon, M. Corn, A. Seaton, and J. B. L. Gee. 1990. Asbestos: scientific developments and implications for public policy. Science 247:294–301. 37. Morrow, J. D., B. Frei, A. W. Longmire, J. M. Gaziano, S. M. Lynch, Y. Shyr, W. E. Strauss, J. A. Oates, and L. J. Roberts. 1995. Increase in circulating products of lipid peroxidation (F2-isoprostanes) in smokers: smoking as a cause of oxidative damage. N. Engl. J. Med. 4:1198–1203. 38. Fung, H., Y. W. Kow, B. Van Houten, and B. T. Mossman. 1997. Patterns of 8-hydroxydeoxyguanosine formation in DNA and indications of oxidative

674

39.

40. 41. 42. 43.

44. 45. 46.

47. 48.

AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL. 20 1999

stress in rat and human pleural mesothelial cells after exposure to crocidolite asbestos. Carcinogenesis 18:825–832. Priemé, H., S. Loft, M. Klarlund, K. Grønbæk, P. Tønnesen, and H. E. Poulsen. 1998. Effect of smoking cessation on oxidative DNA modification estimated by 8-oxo-7,8-dihydro-29-deoxyguanosine excretion. Carcinogenesis 19:347–351. Cheng, K. C., D. S. Cahill, H. Kasai, S. Nishimura, and L. A. Loeb. 1992. 8-Hydroxyguanine, an abundant form of oxidative DNA damage, causes G→T and A→C substitutions. J. Biol. Chem. 267:166–172. Hussain, S. P., F. Aguilar, P. Amstad, and P. Cerutti. 1994. Oxy-radical induced mutagenesis of hotspot codons 248 and 249 of the human p53 gene. Oncogene 9:2277–2281. Bérubé, K. A., T. R. Quinlan, H. Fung, J. Magae, P. Vacek, D. J. Taatjes, and B. T. Mossman. 1996. Apoptosis is observed in mesothelial cells after exposure to crocidolite asbestos. Am. J. Respir. Cell Mol. Biol. 15:141–147. Goldberg, J. L., C. L. Zanella, Y. M. W. Janssen, C. R. Timblin, L. A. Jiminez, P. Vacek, D. J. Taatjes, and B. T. Mossman. 1997. Novel cell imaging techniques show induction of apoptosis and proliferation in mesothelial cells by asbestos. Am. J. Respir. Cell Mol. Biol. 17:265–271. Levine, A. J. 1997. P53, the cellular gatekeeper for growth and division. Cell 88:323–331. Janssen, Y. M. W., N. H. Heintz, and B. T. Mossman. 1995. Induction of c-fos and c-jun proto-oncogene expression by asbestos is ameliorated by N-acetyl-L-cysteine in mesothelial cells. Cancer Res. 55:2085–2089. Jiminez, L. A., C. Zanella, H. Fung, Y. M. W. Janssen, P. Vacek, C. Charland, J. Goldberg, and B. T. Mossman. 1997. Role of extracellular signalregulated protein kinases in apoptosis by asbestos and H2O2. Am. J. Physiol. 273:L1029–L1035. Simeonova, P. P., W. Toriumi, C. Kommineni, M. Erkan, A. E. Munson, W. N. Rom, and M. I. Luster. 1997. Molecular regulation of IL-6 activation by asbestos in lung epithelial cells. J. Immunol. 159:3921–3928. Cote, R. J., S. C. Jhanwar, S. Novick, and A. Pellicer. 1991. Genetic alterations of the p53 gene are a feature of malignant mesotheliomas. Cancer

Res. 51:5410–5416. 49. Metcalf R. A., J. A. Welsh, W. P. Bennett, M. B. Seddon, T. A. Lehman, K. Pelin, K. Linnainmaa, L. Tammilehto, K. Mattson, B. I. Gerwin, and C. C. Harris. 1992. p53 and Kirsten-ras mutations in human mesothelioma cell lines. Cancer Res. 52:2610–2615. 50. Slebos, R. J. C., R. E. Kibbelaar, O. Dalesio, A. Kooistra, J. Stam, C. J. L. M. Meijer, S. S. Wagenaar, R. G. J. R. A. Vanderschueren, N. van Znadwijk, W. J. Mooi, J. L. Bos, and S. Rodenhuis. 1990. K-ras oncogene activation as a prognostic marker in adenocarcinoma of the lung. N. Engl. J. Med. 323:561–565. 51. Mills, N. E., C. L. Fishman, W. N. Rom, N. Dubin, and D. R. Jacobson. 1995. Increased prevalence of K-ras oncogene mutations in lung adenocarcinoma. Cancer Res. 55:1444–1447. 52. Mills, N. E., C. L. Fishman, J. Scholes, S. E. Anderson, W. N. Rom, and D. R. Jacobson. 1995. Detection of K-ras oncogene mutations in bronchoalveolar lavage fluid for lung cancer diagnosis. J. Natl. Cancer Inst. 87: 1056–1060. 53. Mollo, F., E. Pira, G. Piolatto, D. Bellis, P. Burlo, A. Andreozzi, S. Bontempi, and E. Negri. 1995. Lung adenocarcinoma and indicators of asbestos exposure. Int. J. Cancer 60:289–293. 54. Raffn, E., E. Villadsen, G. Engholm, and E. Lynge. 1996. Lung cancer in asbestos cement workers in Denmark. Occup. Environ. Med. 53:399–402. 55. Churg, A. 1985. Lung cancer cell type and asbestos exposure. JAMA 253: 2984–2985. 56. Meurman, L. O., E. Pukkala, and M. Hakama. 1994. Cancer incidence among anthophyllite asbestos quarry workers in Finland. Occup. Environ. Med. 51:421–425. 57. De Klerk, N., W. Musk, J. Eccles, J. Hansen, and M. Hobbs. 1996. Exposure to crocidolite and the incidence of different histological types of lung cancer. Occup. Environ. Med. 53:157–159. 58. Karjalainen, A., E. Pukkala, T. Kauppinen, and T. Partanen. 1998. Incidence of cancer among Finnish patients with asbestos-related pulmonary or pleural fibrosis. Cancer Causes Control (In press)