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Carcinogenesis, Vol. 21, No. 6, 1263-1265, June 2000
© 2000 Oxford University Press

Short Communications

Mice deficient in the nucleotide excision repair gene XPA have elevated sensitivity to benzo[a]pyrene induction of lung tumors

Fumio Ide, Naoko Iida, Yoko Nakatsuru, Hideaki Oda, Kiyoji Tanaka1 and Takatoshi Ishikawa2

Department of Molecular Pathology, Graduate School of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033 and
1 Division of Cellular Genetics, Institute for Molecular and Cellular Biology, Osaka University, 1-3 Yamadaoka, Suita, Osaka 565, Japan


   Abstract
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 Abstract
Introduction
 References
 
This study is focused on chemical induction of lung tumors in xeroderma pigmentosum group A gene (XPA)-deficient mice to clarify the role of nucleotide excision repair (NER) in internal organs. Six-week-old female XPA–/–, XPA+/– and XPA+/+ mice were instilled intratracheally with benzo[a] pyrene (B[a]P). A total of 68 surviving XPA mice treated with B[a]P were examined at month 16. The pulmonary adenoma incidence in XPA–/– mice was significantly higher than that in XPA+/+ mice (71 versus 35%). Similarly, tumor multiplicity was elevated and, in addition, only XPA–/– mice had lung carcinomas. These results provide the first evidence that a deficiency in the NER gene XPA leads to enhanced tumorigenesis in the lung after exposure to B[a]P.

Abbreviations: B[a]P, benzo[a]pyrene; NER, nucleotide excision repair; XP, xeroderma pigmentosum; XPA, XP group A.


   Introduction
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 Abstract
 Introduction
References
 
Nucleotide excision repair (NER) is considered to be a major DNA repair mechanism in mammalian cells, involved in the removal of a wide spectrum of bulky DNA adducts modifying the DNA double helix conformation (1–6). Heritable mutations in NER genes cause cancer-prone human disorders, such as xeroderma pigmentosum (XP) (4,6). Thus it has been extensively documented that XP patients are highly sensitive to sunlight, with a 1000- to 2000-fold increased risk of developing skin cancer (6–11). In addition, epidemiological data point to a 10- to 20-fold increase in internal neoplasms in XP patients, compared with normal individuals (6–9). The question is why there is no evidence of a large increase in mortality from internal neoplasms in XP patients. One possibility is that commonly fatal cancers may not be caused by the kind of DNA lesions that produce neoplasia in XP patients (6,7). Alternatively, they might die from skin cancer before developing internal malignancies.

Among seven different genetic complementation groups in XP, XP group A (XPA) is the most frequently found form which generally displays a severe clinical phenotype (6,8,9). Recently, mice deficient in the NER gene XPA have been generated by gene targeting in embryonic stem cells (12,13). We have already demonstrated that XPA–/– mice exhibit an increased susceptibility to UVB and 7,12-dimethylbenz[a]anthracene induction of skin tumors (12). Despite a difference in the strain background, De Vries et al. (13) obtained similar results. With respect to skin carcinogenesis, these mice, therefore, mimic the phenotype of XPA patients. There have been only a few indications of the importance of NER in protection against chemical carcinogenesis in internal organs of XPA-deficient mice (14). To investigate the effect of NER deficiency on induction of lung tumors, we have exposed XPA-deficient mice to the environmental carcinogen benzo[a] pyrene (B[a]P). Our data provide compelling evidence for an increased sensitivity of XPA–/– mice to chemical induction of internal tumors.

Characterization of the XPA-deficient mice used in the present study has been described in detail previously (12). These mice were housed in a controlled environment at 23°C and fed on NMF diet (Oriental Yeast Co. Ltd, Tokyo, Japan) and sterilized tap water ad libitum. All three genotypes, –/–, +/– and +/+, were determined by PCR using tail tip DNA samples as described earlier (12). At the age of 6 weeks, 30 female mice of each genotype were instilled intratracheally with 0.1 mg of B[a]P (Tokyo Kasei Kogyo, Japan) suspended in 0.02 ml of gelatin/physiological saline under pentobarbital anesthesia once a week for 4 weeks. Five mice of each genotype were treated in the same way with saline as controls. Surviving mice were killed at month 16 of the experiment. The numbers of superficially evident individual lung tumors >0.5 mm in diameter were counted and then the lungs were inflated with 10% buffered formalin. Each lobe of the lungs was completely cut into 2 mm thick sections and embedded in paraffin. Diagnosis was carried out after staining with hematoxylin and eosin using established criteria (15). The statistical significance of differences in numerical data was tested with the {chi}2 test and Student's t-test.

A total of 83 XPA mice including 15 control mice were killed at the end of the experiment in order to assess lung tumor development. Twenty-two mice treated with B[a]P died during the instillation due to suffocation. Since our XPA-deficient mice have a background of a low incidence of spontaneous development of tumors (unpublished data) and the control mice developed very few lesions, primarily alveolar cell hyperplasias, the tumors analyzed in the B[a]P-treated group were deemed to have been caused by B[a]P and not spontaneous. Data for lesion development are summarized in Table IGo. Lung adenomas developed in 71% of XPA–/–, 41% of XPA+/– and 35% of XPA+/+ mice. The tumor incidence in XPA–/– mice was significantly higher than that in XPA+/+ mice (P < 0.05). Tumor multiplicity, with mean ± SE values of 1.4 ± 0.3 for XPA–/–, 0.8 ± 0.2 in XPA+/– and 0.4 ± 0.1 in XPA+/+ mice, was similarly elevated (P < 0.05). Another interesting finding in XPA–/– mice was the presence of lung adenocarcinomas, although of low frequency (10%). Complete autopsy examination also revealed one hepatocellular carcinoma and one ileocecal lymphoma in XPA–/– mice and two splenic lymphomas in XPA+/– mice.


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  		Table I. Development of B[a]P-induced lung tumors in XPA-deficient mice
 
It is well established that XP mice are highly susceptible to UVB induction of skin cancer (12,13,16). The present carcinogenicity study clearly demonstrates that XPA–/– mice are also predisposed to neoplastic change in the lung induced by B[a]P. B[a]P, representative of a multitude of polycyclic aromatic hydrocarbons found widely in the environment, is predominantly metabolized to the reactive form (+)-B[a]P-7,8-diol-9,10-epoxide (17). This intermediate metabolite causes adducts at the N2 position of guanine (17,18). Since primary human XPA fibroblasts are defective in removal of these adducts, the bulky DNA lesions induced by B[a]P are full substrates for NER in mammalian cells (19–21). Concerning the B[a]P-induced internal tumors in XPA-deficient mice, other significant observations have been reported (22–25). De Vries et al. (22) showed that oral gavage with B[a]P resulted in T cell lymphoma in 50% of XPA–/– and 10% of XPA+/+ mice, respectively. Tumors of the lung and forestomach also developed, although at much lower incidences. Their XPA–/– mice showed an increase in Hprt mutant frequency in the spleen but no significant differences in mutant frequencies induced by B[a]P at the lacZ locus in the lung (23,24). Van Oostrom et al., of the same group, also demonstrated that XPA–/– p53–/– double mutant mice have an increased susceptibility to lymphoma (25), but this type of malignancy is not a representative cancer in internal tissues associated with exposure to environmental carcinogens.

Recent studies have demonstrated that XP group C-deficient mice, which are also defective in NER, are predisposed to the development of lung tumors after exposure to 2-acetylaminofluorene (26). This is in keeping with a higher incidence of B[a]P-induced lung tumors in our XPA–/– mice. A study in our laboratory using aflatoxin B1, an important hepatocarcinogenic and toxic mycotoxin (27), showed that the frequency of liver tumors is significantly higher in XPA–/– mice of the same genetic background than in XPA+/+ mice (unpublished data). The preliminary results from our continuing experiments with 4-nitroquinoline 1-oxide (oral administration) indicate that XPA-deficient mice are highly susceptible to developing tongue cancer. These data for XP mice combined with those for XP patients suggest a biological significance of the NER pathway in prevention of cancer in internal organs. Accordingly, in XP patients the elevated occurrence of internal tumors might be closely related to exposure to environmental genotoxic agents and to endogenous oxidative DNA-damaging species which produce lesions which are substrates for NER (28,29). However, it should be borne in mind that NER is not responsible for repair of all DNA damage. It has yet to be resolved how XPA-deficient mice respond to non-genotoxic carcinogens and to genotoxic carcinogens that cause DNA lesions not repaired by NER. Nevertheless, we consider that XPA-deficient mice are potentially useful as a model system for the study of internal tissue carcinogenesis in XP patients.


   Notes
 
2 To whom correspondence should be addressed Back


   Acknowledgments
 
This work was supported by Grants-in-Aid for Scientific Research on Priority Areas from the Ministry of Education, Science and Culture, the Ministry of Health and Welfare of Japan and the Smoking Research Foundation.


   References
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Received December 23, 1999; revised February 8, 2000; accepted February 15, 2000.


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