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[Cancer Research 61, 6679-6681, September 15, 2001]
© 2001 American Association for Cancer Research

Advances in Brief

Lung Tumor KRAS and TP53 Mutations in Nonsmokers Reflect Exposure to PAH-Rich Coal Combustion Emissions1

David M. DeMarini2, Stefano Landi, Defa Tian, Nancy M. Hanley, Xueming Li, Fuding Hu, Barbara C. Roop, Marc J. Mass, Phouthone Keohavong, Weimin Gao, Magali Olivier, Pierre Hainaut and Judy L. Mumford

Environmental Carcinogenesis Division (MD-68) [D. M. D., S. L., N. M. H., B. C. R., M. J. M.], and Human Studies Division [J. L. M.], United States Environmental Protection Agency, Research Triangle Park, North Carolina 27711; Program in Lung Biology, University of North Carolina, Chapel Hill, North Carolina 27599 [D. T.]; Institute of Environmental Health and Engineering, Chinese Academy of Preventive Medicine, Beijing, China [X. L.]; Yunnan Provincial People’s Hospital, Kunming, Yunnan, China [F. H.]; Department of Environmental and Occupational Health and Pittsburgh Cancer Institute, University of Pittsburgh, Pittsburgh, Pennsylvania 15261 [P. K., W. G.]; and International Agency for Research on Cancer (WHO), 150 Cours Albert Thomas, 69372 Lyon Cedex, France [M. O., P. H.]


   ABSTRACT
Top
 ABSTRACT
Introduction
 Materials and Methods
 Results and Discussion
 REFERENCES
 
We determined the TP53 and codon 12 KRAS mutations in lung tumors from 24 nonsmokers whose tumors were associated with exposure to smoky coal. Among any tumors studied previously, these showed the highest percentage of mutations that (a) were G -> T transversions at either KRAS (86%) or TP53 (76%), (b) clustered at the G-rich codons 153–158 of TP53 (33%), and (c) had 100% of the guanines of the G -> T transversions on the nontranscribed strand. This mutation spectrum is consistent with an exposure to polycyclic aromatic hydrocarbons, which are the primary component of the smoky coal emissions. These results show that mutations in the TP53 and KRAS genes can reflect a specific environmental exposure.


   Introduction
Top
 ABSTRACT
 Introduction
Materials and Methods
 Results and Discussion
 REFERENCES
 
A recent report (1) has argued that TP53 mutations in smoking-associated lung tumors are not induced by mutagens in cigarette smoke but are preexisting mutations selected by physiological, nongenotoxic stress. This report has been noted in the medical press (2) , and the validity of inferring environmental exposure from tumor gene mutations has been questioned (1 , 3) . Subsequent analysis by others (4) refutes this position. In this study, we report new data based on an exposure different from cigarette smoke showing that the mutation spectrum in a tumor can, in concert with selection, reflect the exposure linked epidemiologically to that tumor. We demonstrate this by identifying and comparing the TP53 and KRAS mutation spectrum of lung tumors from nonsmokers exposed to polycyclic aromatic hydrocarbon-rich coal emissions to the mutation spectrum of smokers or unexposed nonsmokers.


   Materials and Methods
Top
 ABSTRACT
 Introduction
 Materials and Methods
Results and Discussion
 REFERENCES
 
Lung tumors from 24 nonsmoking women from Xuan Wei County, Yunnan Province, China were obtained embedded in paraffin. These women used smoky coal in their homes, which did not have chimneys. The mean age (± SD) of these women at time of surgery was 48.5 ± 8.8 years, ranging from 30 to 63, with a median age of 50. The percentage of p53-positive cells in the tumor and the intensity of staining were determined by the method described previously (5) . KRAS mutations were determined by denaturing gradient gel electrophoresis by the method described previously (6) . P53 mutations were determined by performing a multiplex PCR for exons 4–9 and then sequencing the amplified exons in both directions by automated sequencing (7) . All of the presumptive mutations were confirmed by repeated sequence analysis. {chi}2 analyses were performed where appropriate.


   Results and Discussion
Top
 ABSTRACT
 Introduction
 Materials and Methods
 Results and Discussion
REFERENCES
 
Almost all (>99%) of the women in Xuan Wei County, Yunnan Province, China are nonsmokers (8) , but they have the highest lung cancer mortality rate in China, 25.3/100,000, which is 8x the national average for females. Their cancers are associated with use of smoky coal, a low-sulfur (0.2%) medium-volatile bituminous coal used for cooking and heating in homes without chimneys (8 , 9) . These emissions contain 81% organic matter, of which 43% is PAH (10) .3 These PAHs account for 61% of the mutagenicity (10) , whereas 58% of the mutagenicity of cigarette smoke is in the aromatic amine-rich basic fraction (11) . Smoky coal emissions are ~1000x more carcinogenic than cigarette smoke in a murine skin-tumor assay, presumably because of their high concentration of PAHs (12) .

Consequently, nonsmokers exposed to these emissions inhale 30x more benzo(a)pyrene that do smokers (13) , and their urine has high levels of PAH metabolites (14) and 600x more benzo(a)pyrene-adducted guanine than does the urine of smokers (13) . Smoky coal-associated lung cancer risk is associated with the GSTM1-null genotype, consistent with the ability of the GSTM1 enzyme to detoxify PAHs (9) . Smoky coal exposure is associated with high levels of TP53 protein accumulation in exfoliated lung cells, suggesting that TP53 mutation may be a critical feature of smoky coal-associated lung cancer (15) . Considering all of the above, nonsmoking women in Xuan Wei with this extreme PAH exposure provided an ideal population to examine whether mutations in tumor genes reflect such an exposure.

Among the 24 women studied here, 54% had bronchioloalveolar adenocarcinoma and 46% had acinar adenocarcinoma (Table 1)Citation . Of the tumors, 29% were mutant at codon 12 of KRAS, with 86% of the mutations being G -> T transversions. All of the 24 tumors stained positive for TP53 protein accumulation, and mutation analysis showed that 71% of the tumors contained base substitutions, with 17% having two TP53 mutations, 21% having mutations in both KRAS and TP53, and only 21% having no detected mutations at either locus (Table 1)Citation .


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  		Table 1 Characteristics of lung tumors

 
The differing chemical compositions of smoky coal combustion emissions and cigarette smoke were reflected in their respective mutation spectra (Table 2)Citation . Of the KRAS mutations, 86% were G -> T transversions in smoky coal-associated tumors, whereas only 66% were among smokers. Of the TP53 mutations, 76% in smoky coal-associated tumors were G -> T transversions, compared with only 29% or 11% among smokers or nonsmokers, respectively. {chi}2 analysis for G -> T transversions in smoky coal versus smokers, versus Asians, and versus nonsmokers gave Ps of 0.09, 0.04, and 0.0003, respectively. The TP53 database contained an insufficient number of Chinese female cigarette smokers to permit a comparison with this subgroup. Although exposure to smoky coal emissions is associated more with adenocarcinomas, and smoking is associated more with squamous-cell carcinomas (5) , the high frequency of G -> T transversions among smoky coal-exposed individuals was not attributable to tumor type because the frequency of such transversions in adenocarcinomas in the IARC TP53 Mutation Database is only 29% (4) . Furthermore, 33% of the mutations in the TP53 gene from smoky coal-associated tumors cluster within the GC-rich region of codons 153–158, whereas only ~9% of the mutations in smokers or nonsmokers (and only 3% in nonsmoking-related tumors) are in this region. This site specificity and high frequency of G -> T transversions are consistent with high PAH exposure and the fact that most PAHs produce DNA damage primarily at guanines (17) .


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  		Table 2 Mutational characteristics of lung tumors

 
Smoky coal-associated TP53 mutations also exhibit an extreme strand bias, such that 100% of the guanines of the G -> T transversions are on the NTS (Table 2)Citation . This suggests the presence of exogenous damage on the NTS that is not repaired by transcription-coupled repair. On the other hand, C -> T transitions at CpG sites are considered to result from an endogenous mutational mechanism, and their frequency is only ~10% in tumors from smoky coal-exposed subjects and smokers (Table 2)Citation . Thus, most mutations in smoky coal-exposed individuals and smokers appear to result from unrepaired, exogenous damage on the NTS rather than resulting from DNA damage that is endogenous in origin.

The three mutational hot spots in the tumors associated with exposure to smoky coal emissions coincide with a hot spot for PAH adducts (codon 154), a hot spot for cigarette smoke-associated mutations (codon 249), and a hot spot for both events (codon 273; Table 3Citation ). Although bearing some similarity to the cigarette smoke-associated tumor spectrum (codons 249 and 273), the smoky coal mutation spectrum is also distinctly different, having a cluster of mutations at codon 154, which is not a hot spot for cigarette smoke-associated mutations.


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  		Table 3 Hot spots in TP53 of PAH adducts and mutations associated with exposure to cigarette smoke or smoky coal emissions

 
Along with studies on environmental tobacco smoke (18 , 19) and radon (20) , this study on smoky coal presents a unique mutation spectrum in lung tumors from nonsmokers whose cancers are linked epidemiologically to a well-characterized exposure. As such, our results and other analyses (4) support the view that mutation spectra in tumor genes reflect, in concert with selection, the primary DNA damage induced by mutagenic exposures linked epidemiologically to those tumors.


   ACKNOWLEDGMENTS
 
We thank Tao Chen (U.S. EPA, Research Triangle Park, NC) and Daniel Shaughnessy (University of North Carolina, Chapel Hill, NC) for their assistance with DNA sequence analysis. This project was performed under the China-United States Protocol, Annex 1, II.A for Scientific and Technical Cooperation in the Field of Environmental Protection. The research protocol was approved by a United States Environmental Protection Agency Human Subjects Research Review Official. This manuscript has been reviewed by the National Health and Environmental Effects Research Laboratory, United States Environmental Protection Agency and approved for publication. Approval does not signify that the contents necessarily reflect the views and policies of the Agency, nor does mention of trade names or commercial products constitute endorsement or recommendation for use.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Partially supported by a Grant from the American Cancer Society (RPG-99-161-01-CNE; to P. K. and W. G.). Back

2 To whom requests for reprints should be addressed, at Environmental Carcinogenesis Division (MD-68), United States Environmental Protection Agency, Research Triangle Park, North Carolina 27711. E-mail: demarini.david{at}epa.gov Back

3 The abbreviations used are: PAH, polycyclic aromatic hydrocarbon; NTS, nontranscribed strand. Back

Received 5/31/01. Accepted 8/ 1/01.


   REFERENCES
Top
 ABSTRACT
 Introduction
 Materials and Methods
 Results and Discussion
 REFERENCES
 

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