Betel nut and susceptibility to cancer

Environmental Factors, Genes, and the Development of Human Cancers

Volumn , Issue , 2010, Pages 401-428

  Sharan, Rajesh N ^a   Choudhury, Yashmin ^a  

^a NORTH EASTERN HILL UNIVERSITY   (India)

Author keywords

Betel nut specific nitrosamine derivatives and alkaloids; Mutagenic and genotoxic effects; Susceptibility to oral cancer; Tumor suppressor genes

Indexed keywords

EID: 84900056598     PISSN: None     EISSN: None     Source Type: Book    
DOI: 10.1007/978-1-4419-6752-7_15     Document Type: Chapter

References (97)

1. Balachandran, B., Sharan, R. N. 1995. Induction of mutations by different extracts of betel nut and radiation: Their implication in carcinogenesis. In Radiation Research 1895-1995, eds. U. Hagen, H. Jung, and C. Streffer, pp 165, vol. 1, Würzburg: Universitätsdruckerei H. Stürtz AG.
2. Bartsch, H., Hietanen, E. 1996. The role of individual susceptibility in cancer burden related to environmental exposure. Environ Health Perspect 104:569-577.
3. + ions. Mol. Med. Rep. 1:435-441.
4. Boucher, B. J., Mannan, N. 2002. Metabolic effects of the consumption of Areca catechu. Addict. Biol. 7:103-110.
5. Chang, M. C., Ho, Y. S., Lee, P. H., Chan, C. P., Lee, J. J., Hahn, L. J., Wang, Y. J., Jeng, J. H. 2001d. Areca nut extract and arecoline induced the cell cycle arrest but not apoptosis of cultured oral KB epithelial cells: association of glutathione, reactive oxygen species and mitochondrial membrane potential. Carcinogenesis 22:1527-1535.
6. Chang, Y. C., Hu, C. C., Lii, C. K., Tai, K. W., Yang, S. H., Chou, M. Y. 2001b. Cytotoxicity and arecoline mechanisms in human gingival fibroblasts in vitro. Clin. Oral Investig. 5:51-56.
7. Chang, Y. C., Hu, C. C., Tseng, T. H., Tai, K. W., Lii, C. K., Chou, M. Y. 2001a. Synergistic effects of nicotine on arecoline-induced cytotoxicity in human buccal mucosal fibroblasts. J. Oral Pathol. Med. 30:458-464.
8. Chang, B., Liao, M., Kuo, M. Y., Chen, C. 2001c. Developmental toxicity of arecoline, the major alkaloid in betel nuts, in zebrafish embryos. Birth Defects Res. 70:28-36.
9. Chang, M. C., Wu, H. L., Lee, J. J., Lee, P. H., Chang, H. H., Hahn, L. J., Lin, B. R., Chen, Y. J., Jeng, J. H. 2004. The induction of prostaglandin E2 production, interleukin-6 production, cell cycle arrest, and cytotoxicity in primary oral keratinocytes and KB cancer cells by areca nut ingredients is differentially regulated by MEK/ERK activation. J Biol Chem 279:50676-50683.
10. Chen, C. L., Chi, C. W., Chang, K. W., Liu, T. Y. 1999. Safrole-like DNA adducts in oral tissue from oral cancer patients with a betel quid chewing history. Carcinogenesis 20:2331-2334.
11. Chiang, C., Chang, M., Lee, J., Chang, J. Y., Lee, P., Hahn, L., Jeng, J. 2004. Hamsters chewing betel quid or areca nut directly show a decrease in body weight and survival rates with concomitant epithelial hyperplasia of cheek pouch. Oral Oncol. 40:720-727.
12. Chiang, S., Jiang, S., Wang, Y., Chiang, H., Chen, P., Tu, H., Ho, K., Tsai, Y., Chang, I., Ko, Y. 2007. Characterization of arecoline-induced effects on cytotoxicity in normal human gingival fibroblasts by global gene expression profiling. Toxicol Sci 100:66-74.
13. Chiba, I., Muthumala, M., Yamazaki, Y., Zaman, A. U., Iizuka, T., Amemiya, A., Shibata, T., Kashiwazaki, H., Sugiura, C., Fukuda, H. 1998. Characteristics of mutations in the p53 gene of oral squamous-cell carcinomas associated with betel-quid chewing in Sri Lanka. Int J Cancer 77:839-842.
14. Chiu, C. J., Chang, C. P., Hahn, L. J., Hsieh, L. L., Chen, C. J. 2002. Interaction of collagen-related genes and susceptibility to betel quid-induced oral submucous fibrosis. Cancer Epidemiol. Biomarkers. Prev. 11:646-653.
15. Chiu, C. J., Chiang, C. P., Chang, M. L., Chen, H. M., Hahn, L. J., Hsieh, L. L., Kuo, Y. S., Chen, C. J. 2001. Association between genetic polymorphism of tumor necrosis factor-alpha and risk of oral submucous fibrosis, a pre-cancerous condition of oral cancer. J. Dent. Res. 80:2055-2059.
16. Choudhury, Y., Sharan, R. N. 2009. Altered p53 response and enhanced transgenerational transmission of carcinogenic risk upon exposure of mice to betel nut. Environ. Toxicol. Pharmacol. 27:127-138.
17. Choudhury, Y., Sharan, R. N. 2010. Altered BRCA1 and BRCA2 response and mutation of BRCA1 gene m mice exposed chronically and transgenerationally to aqueous extract of betel nut. Environ. Toxicol. Pharmacol. (in communication)
18. Chung, K. T., Wong, T. Y., Wei, C. I., Huang, Y. W., Lin, Y. 1998. Tannins and human health: a review. Crit. Rev. Food Sci. Nutr. 38:421-464.
19. Cressman, V. L., Backlund, D. C., Avrutskaya, A., Leadon, S. A., Godfrey, V., Koller, B. H. 1999. Growth retardation, DNA repair defects, and lack of spermatogenesis in BRCA1-deficient mice. Mol Cell Biol 19:7061-7075.
20. Deb, S., Chatterjee, A. 1998. Influence of buthionine sulfoximine and reduced glutathione on arecoline-induced chromosomal damage and sister chromatid exchange in mouse bone marrow cells in vivo. Mutagenesis 13:243-248.
21. Deng, J. F., Ger, J., Tsai, W. J., Kao, W. F., Yang, C. C. 2001. Acute toxicities of betel nut: rare but probably overlooked events. J Toxicol Clin Toxicol 39:355-360.
22. Desai, S. S., Ghaisas, S. D., Jakhi, S. D., Bhide, S. V. 1996. Cytogenetic damage in exfoliated oral mucosal cells and circulating lymphocytes of patients suffering from precancerous oral lesions. Cancer Lett. 109:9-14.
23. Goan, Y. G., Chang, H. C., Hsu, H. K., Chou, Y. P., Cheng, J. T. 2005. Risk of p53 gene mutation in esophageal squamous cell carcinoma and habit of betel quid chewing in Taiwanese. Cancer Sci. 96:758-765.
24. Harris, C. C., Hollstein, M. 1993. Clinical implications of the p53 tumor-suppressor gene. N Engl J Med 329:1318-1327.
25. Hollstein, M., Sidransky, D., Vogelstein, B., Harris, C. C. 1991. P53 mutation in human cancer. Science 253:49-53.
26. Hsieh, L. L., Wang, P. F., Chen, I. H., Liao, C. T., Wang, H. M., Chen, M. C., Chang, J. T., Cheng, A. J. 2001. Characterisics of mutations of the p53 gene in oral squamous cell carcinoma associated with betel quid chewing and cigarette smoking in Taiwanese. Carcinogenesis 22:1497-1503.
27. Hsu, H. J., Chang, K. L., Yang, Y. H., Shieh, T. Y. 2001. The effects of arecoline on the release of cytokines using cultured peripheral blood mononuclear cells from patients with oral mucous diseases. Kaohsiung J Med Sci 17:175-182.
28. Hung, S., Cheng, Y., Peng, J., Chang, L., Liu, T., Chen, Y. 2005. Inhibitory effects of areca nut extracts on phagocytosis of Actinobacillus actinomycetemcomitans ATCC 33384 by neutrophils. J. Periodontol. 76:373-379.
29. IARC. 1985. International Agency for Research on Cancer (IARC) - Tobacco habits other than smoking: Betel quid and Areca-nut chewing and some related nitrosamines. IARC Monogr Eval Carcinog Risks Hum 37:263, Lyon: IARC.
30. IARC. 2004. International Agency for Research on Cancer (IARC) - Summaries & Evaluations: Betel-quid and areca-nut chewing. IARC Monogr Eval Carcinog Risk Chem Hum 85:39, Lyon: IARC.
31. Jeng, J. H., Chang, M. C., Hahn, L. J. 2001. Role of areca nut in betel quid-associated chemical carcinogenesis: current awareness and future perspectives. Oral Oncol. 37:477-492.
32. Jeng, J. H., Kuo, M. L., Hahn, L. J., Kuo, M. Y. 1994. Genotoxic and non-genotoxic effects of betel quid ingredients on oral mucosal fibroblasts in vitro. J. Dental Res. 73:1043-1049.
33. Jeng, J. H., Lan, W. H., Hahn, L. J., Hsieh, C. C., Kuo, M. Y. 1996. Inhibition of the migration, attachment, spreading, growth and collagen synthesis of human gingival fibroblasts by arecoline, a major areca alkaloid, in vitro. J. Oral Pathol. Med. 25:371-375.
34. Jeng, J. H., Tsai, C. L., Hahn, L. J., Yang, P. J., Kuo, Y. S., Kuo, M. Y. 1999. Arecoline cytotoxicity on human oral mucosal fibroblasts related to cellular thiol and esterase activities. Food Chem Toxicol 37:751-756.
35. Jeng, J. H., Wang, Y. J., Chiang, B. L., Lee, P. H., Chan, C. P., Ho, Y. S., Wang, T. M., Lee, J. J., Hahn, L. J., Chang, M. C. 2003. Roles of keratinocyte inflammation in oral cancer: regulating the prostaglandin E2, interleukin-6 and TNF-a production of oral epithelial cells by areca nut extract and arecoline. Carcinogenesis 24:1301-1315.
36. Kannan, K., Munirajan, A. K., Krishnamurthy, J. 1999. Low incidence of p53 mutation in betel quid and tobacco chewing-associated oral squamous carcinoma from India. Int J Oncol 15:1133-1136.
37. Kao, S. Y., Wu, H. C., Lin, S. C., Yap, S. K., Chang, C. S., Wong, Y. K., Chi, L. Y., Liu, T. Y. 2002. Genetic polymorphism of cytochrome P4501A1 and susceptibility to oral squamous cell carcinoma and oral precancer lesions associated with smoking/betel use. J. Oral Pathol. Med. 31:505-511.
38. Kato, K., Hara, A., Kuno, T., Kitaori, N., Huilan, Z., Mori, H., Toida, M., Shibata, T. 2005. Matrix metalloproteinases 2 and 9 in oral squamous cell carcinomas: manifestation and localization of their activity. J. Cancer Res. Clin. Oncol. 131:340-346.
39. Kietthubthew, S., Sriplung, H., Au, W. W. 2001. Genetic and environmental interactions on oral cancer in Southern Thailand. Environ Mol Mutagen 37:111-116.
40. Kinzler, K. W., Vogelstein, B. 1997. Gatekeeprs and caretakers. Nature 386:761-763.
41. Kumpawat, K., Deb, S., Ray, S., Chatterjee, A. 2003. Genotoxic effect of raw betel-nut extract in relation to endogenous glutathione levels and its mechanism of action in mammalian cells. Mutat Res 538:1-12.
42. Kuttan, N. A. A., Rosin, M. P., Ambika, K., Priddy, R. W., Bhakthan, N. M. G., Zhang, L. 1995. High prevalence of expression of p53 oncoprotein in oral carcinomas from India associated with betel and tobacco chewing. Eur J Cancer 31:169-173.
43. Lai, K., Lee, T. 2006. Genetic damage in cultured human keratinocytes stressed by long-term exposure to areca nut extracts. Mutat. Res. Fundam. Mol. Mech. Mutag. 599:66-75.
44. Lane, D. P. 1992. Cancer: p53 guardian of the genome. Nature 358:15-16.
45. Lee, P., Chang, M., Chang, W., Wang, T., Wang, Y., Hahn, L., Ho, Y., Lin, C., Jeng, J. 2006. Prolonged exposure to arecoline arrested human KB epithelial cell growth: Regulatory mechanisms of cell cycle and apoptosis. Toxicology 220:81-89.
46. Lee, H., Yin, P., Yu, T., Chang, Y., Hsu, W., Kao, S., Chi, C., Liu, T., Wei, Y. 2001. Accumulation of mitochondrial DNA deletions in human oral tissues - effects of betel quid chewing and oral cancer. Mutat. Res. Genetic Toxicol. Environ. Mutagen. 493:67-74.
47. Levine, A. J. 1997. P53, the cellular gatekeeper for growth and division. Cell 88:323-331.
48. Lin, Y., Jen, Y., Wang, B., Lee, J., Kang, B. 2005. Epidemiology of oral cavity cancer in Taiwan with emphasis on the role of betel nut chewing. ORL J. Otorhinolaryngol. Relat. Spec. 67:230-236.
49. Lin, S. C., Liu, C. J., Yeh, W. I., Lui, M. T., Chang, K. W., Chang, C. S. 2006. Functional polymorphism in NFKB1 promoter is related to risks of oral squamous cell carcinoma occurring on older male areca (betel) chewers. Cancer Lett. 243:47-54.
50. Liu, T., Chen, C., Chi, C. 1996. Oxidative damage to DNA induced by areca nut extract. Mutat. Res. Genetic Toxicol. 367:5-31.
51. Liu, T. Y., Chi, C. W. 1999. Areca nut extracts stimulate DNA strand breaks by generation of nitric oxide. Toxicol Lett 95:112.
52. Liu, T. Y., Chung, Y. T., Wang, P. F., Chi, C. W., Hsieh, L. L. 2004. Safrole-DNA adducts in human peripheral blood-an association with areca quid chewing and CYP2E1 polymorphisms. Mutat. Res. Genetic Toxicol. Environ. Mutagen. 559:59-66.
53. Liu, S. Y., Lin, M. H., Yang, S. C., Huang, G. C., Chang, L., Chang, S., Yen, C. Y., Chiang, W. F., Kuo, Y. Y., Chen, L. L., Lee, C. H., Liu, Y. C. 2005a. Increased expression of matrix metalloproteinase-2 in oral cells after short-term stimulation and long-term usage of areca quid. J. Formos. Med. Assoc. 104:390-397.
54. Liu, S. Y., Lin, M. H., Yang, S. C., Huang, G. C., Chang, L., Chang, S., Yen, C. Y., Chiang, W. F., Lee, C. H., Kuo, Y. Y., Liu, Y. C. 2005b. Areca quid chewing enhances the expression of salivary matrix metalloproteinase-9. J. Formos. Med. Assoc. 104:113-119.
55. 1 phase arrests and senescence-associated phenotypes in normal human oral keratinocyte. Carcinogenesis 27:1273-1284.
56. Nadeau, G., Boufaied, N., Moisan, A., Lemieux, K. M., Cayanan, C., Monteiro, A. N. A., Gaudreau, L. 2000. BRCA1 can stimulate gene transcription by a unique mechanism. EMBO Rep. 3:260-265.
57. Nair, U., Bartsch, H., Nair, J. 2004. Alert for an epidemic of oral cancer due to use of the betel quid substitutes gutkha and pan masala: a review of agents and causative mechanisms. Mutagenesis 19:251-262.
58. Nair, U., Floyd, R. A., Nair, J., Bussachini, V., Friesen, M., Bartsch, H. 1987. Formation of reactive oxygen species and of 8-hydroxydeoxyguanosine in DNA in vitro with betel quid ingredients. Chem Biol Interact 63:157-169.
59. Nair, U. J., Nair, J., Mathew, B., Bartsch, H. 1999. Glutathione S-transferase M1 and T1 null genotypes as risk factors for oral leukoplakia in ethnic Indian betel quid/tobacco chewers. Carcinogenesis 20:743-748.
60. Nair, J., Ohshima, H., Friesen, M., Croisy, A., Bhide, S. V., Bartsch, H. 1985. Tobacco-specific and betel nut-specific N-nitroso compounds: occurrence in saliva and urine of betel quid chewers and formation in vitro by nitrosation of betel quid. Carcinogenesis 6:295-303.
61. Norton, S. A. 1998. Betel: consumption and consequences. J Am Acad Dermatol 38:81-88.
62. Panigrahi, G. B., Rao, A. R. 1989. Study of the genotoxicity of the total aqueous extract of betel nut and its tannin. Carcinogenesis 7:37-39.
63. Pariat, T., Balachandran, B., Sharan, R. N. 1999. Effects of carcinogen exposure on poly-ADP-ribosylation of HMG Proteins and on chromatin organization. In Recent Aspects of Cellular and Applied Radiobiology, eds. F. H. A. Schneeweiss and R. N. Sharan, pp 158-161, vol. 30, International co-operation bilateral seminars series, Juelich: Forschungszentrum Juelich GmbH.
64. Pariat, T., Sharan, R. N. 1998a. Betel nut and diethylnitrosamine induced conformational changes in the liver HMG proteins in vivo. In Trends in Radiation and Cancer Biology, ed. R. N. Sharan, pp 162-165, vol. 29, International co-operation bilateral seminars series, Juelich: Forschungszentrum Juelich GmbH.
65. Pariat, T., Sharan, R. N. 1998b. Qualitative change in mice liver HMG proteins after low dose chronic administration of aqueous extract of betel nut and diethynitrosamine. Hepatol Res 12:177-185.
66. Pariat, T., Sharan, R. N. 2002. Role of mouse spleen cell HMG proteins and its poly-ADP-ribosylation in betel nut induced carcinogenesis. Indian J Biochem Biophys 39:130-132.
67. Patel, B. P., Shah, S. V., Shukla, S. N., Shah, P. M., Patel, P. S. 2007. Clinical significance of MMP-2 and MMP-9 in patients with oral cancer. Head Neck 29:564-572.
68. Paul, K., Moitra, P. K., Mukherjee, I., Maity, C., Ghosal, S. K. 1999. Teratogenicity of arecoline hydrobromide on developing chick embryos: A preliminary report. Bull Environ Contam Toxicol 62:356-362.
69. Polasa, K., Babu, S., Shenolikar, I. S. 1993. Dose-dependent genotoxic effect of pan masala and areca nut in Salmonella typhimurium assay. Food Chem Toxicol 31:439-442.
70. Rajan, J. V., Wang, M., Marquis, S. T., Chodosh, L. A. 1996. Brca2 is coordinately regulated with Brca1 during proliferation and differentiation in mammary epithelial cells. Proc. Natl. Acad. Sci. (USA) 93:13078-13083.
71. Ralhan, R., Agarwal, S., Nath, N., Mathur, M., Wasylyk, B., Srivastava, A. 2001. Correlation between p53 gene mutations and circulating antibodies in betel- and tobacco-consuming North Indian population. Oral Oncol. 37:243-250.
72. Ramachandran, S., Ramadas, K., Hariharan, R., Rejnish, K. R., Radhakrishna, P. M. 2006. Single nucleotide polymorphisms of DNA repair genes XRCC1 and XPD and its molecular mapping in Indian oral cancer. Oral Oncol. 42:350-362.
73. Ranasinghe, A. W., Warnakulasuriya, K. A. A. S., Johnson, N. W. 1993. Low prevalence of expression of p53 oncoprotein in oral carcinomas from Sri Lanka associated with betel and tobacco chewing. Eur J Cancer 29:147-150.
74. Saikia, J. R., Schneeweiss, F. H. A., Sharan, R. N. 1998. Effects of chronic low-dose arecoline administration on the macromolecular components of bone marrow and spleen cells of mice. Cancer J. 11:94-98.
75. Saikia, J. R., Schneeweiss, F. H. A., Sharan, R. N. 1999a. Chronic low dose arecoline exposure affects DNA, RNA and protein contents and cellular poly-ADP-ribosylation in mice in vivo. In Recent Aspects of Cellular and Applied Radiobiology, eds. F. H. A. Schneeweiss, and R. N. Sharan, pp 149-152, vol. 30, International co-operation bilateral seminars series, Juelich: Forschungszentrum Juelich GmbH.
76. Saikia, J. R., Schneeweiss, F. H. A., Sharan, R. N. 1999b. Arecoline-induced changes of poly-ADP-ribosylation of cellular proteins and its influence on chromatin organization. Cancer Lett. 139:59-65.
77. Sharan, R. N. 1994. Biochemical investigation of carcinogenic potency of betel nut (Kwai) of north-east India. In, Oral Oncology, pp. 190-193, vol. III, New Delhi: MacMillan India Ltd.
78. Sharan, R. N. 1996. Association of betel nut with carcinogenesis - A review. Cancer J. 9:13-19.
79. Sharan, R. N., Wary, K. K. 1992. Study of unscheduled DNA synthesis following exposure of human cells to arecoline and extracts of betel nut in vitro. Mutat Res 278:271-276.
80. Shwe, M., Chiguchi, G., Yamada, S., Nakajima, T., Maung, K. K., Takagi, M., Amagasa, T., Tsuchida, N. 2001. P53 and MDM2 co-expression in tobacco and betel chewing-associated oral squamous cell carcinomas. J. Med. Dent. Sci. 48:113-119.
81. Sinha, A., Rao, A. R. 1985a. Embryotoxicity of betel nuts in mice. Toxicol. 37:315-326.
82. Sinha, A., Rao, A. R. 1985b. Induction of shape abnormality and unscheduled DNA synthesis by arecoline in the germ cells of mice. Mutat Res 158:189-192.
83. Sinha, A., Rao, A. R. 1985c. Transplacental micronucleus inducing ability of arecoline, a betel nut alkaloid in mice. Mutat Res 158:193-194.
84. Spitz, M. R., Bondy, M. L. 1993. Genetic susceptibility to cancer. Cancer. 72:991-995.
85. Stich, H. F., Anders, F. 1989. The involvement of reactive oxygen species in oral cancers of betel quid/tobacco chewers. Mutat Res 214:47-61.
86. Sundqvist, K., Liu, Y., Nair, J., Bartsch, H., Arvidson, K., Grafström, R. C. 1989. Cytotoxic and genotoxic effects of areca nut-related compounds in cultured human buccal epithelial cells. Cancer Res 49:5294-5298.
87. Thomas, S., Brennan, J., Martel, G. 1994. Mutations in the conserved regions of p53 are infrequent in betel-associated oral cancers from Papua New Guinea. Cancer Res 54:3588-3593.
88. Topcu, Z., Chiba, I., Fujieda, M., Shibata, T., Ariyoshi, N., Yamazaki, H., Sevgican, F., Muthumala, M., Kobayahi, H., Kamataki, T. 2002. CYP2A6 gene deletion reduces oral cancer risk in betel quid chewers in Sri Lanka. Carcinogenesis 23:595-598.
89. Trivedy, C. R., Craig, G., Warnakulasuriya, S. 2002. The oral health consequences of chewing areca nut. Addict. Biol. 7:115-125.
90. Tseng, C. 2006. Betel nut chewing is independently associated with urinary albumin excretion rate in type 2 diabetic patients. Diabetes Care 29:462-463.
91. Tu, H. F., Liu, C. J., Chang, C. S., Lui, M. T., Kao, S. Y., Chang, C. P., Liu, T. Y. 2006. The functional (-1171 5A→>6A) polymorphisms of matrix metalloproteinase 3 gene as a risk factor for oral submucous fibrosis among male areca users. J. Oral Pathol. Med. 35:99-103.
92. Tu, H. F., Wu, C. H., Kao, S. Y., Liu, C. J., Liu, T. Y., Lui, M. T. 2007. Functional-1562 C-to-T polymorphism in matrix metalloproteinase-9 (MMP-9) promoter is associated with the risk for oral squamous cell carcinoma in younger male areca users. J. Oral Pathol. Med. 36:409-414.
93. Wang, C. K., Peng, C. H. 1996. The mutagenicities of alkaloids and N-nitrosoguvacoline from betel quid. Mutat Res Environ Mutagen Relat Subj 360:165-171.
94. Warnakulasuriya, S. 2002. Areca nut use: an independent risk factor for oral cancer. Br. Med. J. 324:799-800.
95. Wary, K. K., Sharan, R. N. 1988. Aqueous extract of betel-nut of North-East India induces DNA strand breaks and enhances rate of cell proliferation in vitro. J. Cancer Res. Clin. Oncol 114:579-582.
96. Wary, K. K., Sharan, R. N. 1991. Cytotoxic and cytostatic effects of arecoline and sodium nitrite on human cells in vitro. Int J Cancer 47:396-400.
97. Wen, X., Zhang, Y., Liu, X., Guo, S., Wang, H. 2006. Immune responses in mice to arecoline mediated by lymphocyte muscarinic acetylcholine receptor. Cell Biol. Int. 30:1048-1053.