Inhibition of benzo[a]pyrene-induced mutagenesis by ()-epigallocatechin gallate in the lung of rpsL transgenic mice
Shigeharu Muto,
Tsuyoshi Yokoi,
Yoichi Gondo1,
Motoya Katsuki2,
Yoshiyuki Shioyama3,
Ken-ichi Fujita and
Tetsuya Kamataki4
Laboratory of Drug Metabolism, Graduate School of Pharmaceutical Sciences, Hokkaido University, Sapporo 060-0812,
1 Institute of Medical Sciences, Tokai University, Isehara 259-1100,
2 Institute of Medical Sciences, University of Tokyo, Bunkyo-ku, Tokyo 113-0033 and
3 Division of Molecular and Cellular Biology, Medical Institute of Bioregulation, Kyushu University, Fukuoka 812-0054, Japan
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Abstract
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Epigallocatechin gallate (EGCG) is a major water-soluble component of green tea. The antimutagenic activity of EGCG against benzo[a]pyrene (B[a]P)-induced mutations was assessed by using transgenic mice carrying the rpsL gene as a monitor of mutations. Seven-week-old male mice were given drinking water containing EGCG for 3 weeks. On day 7, mice were treated with a single i.p. injection of B[a]P (500 mg/kg body wt). Two weeks after the injection, the mutations in the rpsL gene were analyzed. B[a]P treatment resulted in an ~4-fold increase of mutation frequency at the rpsL gene in the lung. An ~60% reduction in the B[a]P-induced mutations in the lung was observed when mice were given EGCG at concentrations >0.005%. B[a]P-induced mutations mainly occurred at G:C base-pairs in the several specific nucleotide sequences of the rpsL gene. These were AGG, CGG, CGT, TGG, TGC and GGT: all of them contained a guanine residue. Mutations seen similarly in the human Ki-ras codon 12 or p53 codons 157, 248, and 273 of lung tumor were also found in the rpsL gene, and the mutations were suppressed by the EGCG treatment. In conclusion, the antimutagenic effects of EGCG for B[a]P-induced mutagenesis in vivo suggest that drinking green tea may reduce the tumor-initiating potency of B[a]P in the lung.
Abbreviations: AFB1, aflatoxin B1; B[a]P, benzo[a]pyrene; BPDE, (±)-7ß,8
-dihydroxy-9
,10
-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene; EGCG, ()-epigallocatechin gallate; KM, kanamycin; SM, streptomycin.
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Introduction
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It has been generally accepted that the gene mutations induced by various chemicals are important as an initial event in carcinogenesis (14). Thus, a factor(s) that decreases the mutations can be expected to reduce the incidence of cancer.
Green tea, prepared from the dried leaves of Camellias sincesis, is one of the most widely consumed beverages in the world. Several studies have demonstrated that polyphenols contained in green tea have chemopreventive effects on chemical carcinogenesis in many organs such as the lung (57), forestomach (6,7), liver (8), esophagus (9), duodenum (9) and colon (10) in rodents, and that polyphenols in green tea show antimutagenic activity in vitro (11,12). Among polyphenols, ()-epigallocatechin-3-gallate (EGCG), the major polyphenol component in green tea, is the most effective antimutagen in vitro (11). Wang et al. reported that EGCG significantly inhibited the mutagenicity of B[a]P, aflatoxin B1 (AFB1) and 2-aminofluorene in a mutation assay using Salmonella typhimurium TA100 and TA98 strains (11). Okuda et al. also reported that EGCG strongly inhibited the mutagenicity of the ultimate carcinogenic form of B[a]P, (±)-7ß,8
-dihydroxy-9
,10
-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE), using S.typhimurium TA100 (12). However, the results obtained from in vitro systems do not necessarily reflect physiological functions, considering the distribution of compounds and other factors. Therefore, it was of interest to investigate whether or not polyphenols contained in green tea showed its antimutagenic activity in vivo.
Recently, the rpsL transgenic mouse line with an Escherichia coli shuttle vector has been established as a proficient and practical short-term tool for an in vivo mutagenicity assay (13). In the present study, rpsL transgenic mice were used to evaluate the effects of oral feeding of EGCG on the mutagenicity of B[a]P in vivo. The results of this study clearly show that EGCG prevents the mutation caused by B[a]P in vivo.
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Materials and methods
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Chemicals
B[a]P and EGCG were purchased from Wako Pure Chemical Industries (Tokyo, Japan). All other chemicals were of the highest quality commercially available.
Animals and treatment
Transgenic mice with the background of C57BL/6J mice, carrying ~350 copies of the rpsL gene in the shuttle plasmid pML4, were used. All the transgenic mice were maintained hemizygously. They were bred within transgenic animal facilities in the Faculty of Pharmaceutical Sciences, Hokkaido University. A sterilized and ventilated environment with constant temperature at 23 ± 1°C, a relative humidity of 55 ± 5% and 12 h lightdark cycle was automatically controlled by a central system. Animals were housed in plastic cages with free access to a commercial rodent chow (Clea, Tokyo, Japan) and tap water ad libitum.
Seven-week-old male mice from an rpsL transgenic line were divided into four groups, which were given drinking water containing 0, 0.001, 0.005 or 0.05% EGCG for 3 weeks. On day 7, half of the animals per group were treated with B[a]P at a dose of 500 mg/kg in 5 ml corn oil by a single i.p. injection. The other half of the animals were treated with corn oil. Fourteen days later, mice were killed and lungs were collected. They were quickly frozen in liquid nitrogen and stored at 80°C until analysis.
rpsL transgenic mouse mutagenesis assay
Mutation analysis was performed as described by Gondo et al. (13) with minor modifications. Briefly, genomic DNA was prepared from the lung. Aliquots of 20 µg of isolated genomic DNA were digested with 1.75 U BanII (Takara, Tokyo, Japan) per µg of DNA. To circularize the pML4 plasmids, 10 µg of DNA digested with BanII was treated with 4 Weiss units of T4 DNA ligase (Takara) per µg of DNA in a final volume of 1000 ml. The ligated DNA was resuspended in 1 mM TrisHCl containing 0.1 mM Na2EDTA (pH 8.0) to give a final concentration of 1 µg/µl. The RR1 strain, which is resistant to streptomycin (SM) and sensitive to kanamycin (KM), was transformed with the circularized pML4. Competent E.coli RR1 cells were prepared as described by Gondo et al. (13). A Cell Porator E.coli System (Bio-Rad, CA) was used for electroporation according to the manufacturer's instructions. An aliquot of 100 µl of E.coli cell suspension was mixed with 5 µl of the prepared DNA. The mixture was divided into five portions (21 µl). Each aliquot was loaded into a disposable electroporation chamber (Bio-Rad), and then the electroporation was conducted at 1.8 kV/mm at 4°C. Total mixture was transferred into an SOC medium (5 ml) (14) and incubated at 37°C with vigorous shaking for 160 min. After incubation for 70 min, an aliquot (25 µl) of the SOC culture was plated onto an H plate (0.1% bacto-trypton, 160 mM NaCl, 1.2% agar) containing KM (50 µg/ml) alone to evaluate the total screened number of the rpsL gene. The remainder was plated onto an H plate containing KM (50 µg/ml) and SM (100 µg/ml) to detect E.coli cells carrying a mutated rpsL gene. The mutation frequency was then calculated as the number of mutated rpsL carriers/the total screened number of pML4.
A shuttle plasmid vector pML4 carrying a mutated rpsL gene was extracted from KM- and SM-resistant RR1 cells as described previously (14). An entire rpsL gene was sequenced by ABI fluorescent sequencer model 377A (Perkin-Elmer, CA) with a PRISM DyeDeoxy Terminator Cycle Sequencing kit (Perkin-Elmer, CA). Each mutation was confirmed by duplicate sequence analysis for both strands of the rpsL gene. Primers used for the sequence analysis were complementary to positions from 16 to 4 of the rpsL gene (5'-GTTTACGAAGCAAAAGCTAA-3') and positions from +436 to +455 of the rpsL gene (5'-GGCATGGAAATACTCCGTTG-3'). Mutations in the rpsL gene were analyzed using Genetyx (Software Development, Tokyo, Japan) by aligning the sequence against the reference of a wild-type rpsL gene.
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Results and discussion
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Oral feeding of EGCG did not result in any apparent toxic effects even at a high concentration of EGCG (0.05%). No significant change was observed in food and water consumption and body weight (data not shown). The frequency of spontaneous mutation in the rpsL gene in the lung was 2.7 ± 1.0x 105. A single i.p. injection of B[a]P resulted in ~4-fold increase of mutation frequency (11.7 ± 4.0x105). The B[a]P-induced mutations in the lung were reduced by ~60% when mice were given EGCG at concentrations of 0.005 or 0.05% (Figure 1
). Katiyar et al. reported that oral feeding of green tea and a polyphenolic fraction from green tea during the initiation period significantly suppressed B[a]P-induced lung tumorigenesis in A/J mice (6,7). Green tea and the polyphenolic fraction from green tea contained ~0.08% and 0.12% EGCG, respectively (6,7). A further reduction in mutation frequency was not observed when mice were given water containing EGCG at a concentration >0.005%. Supporting this, Nakagawa et al. reported that the plasma concentration of EGCG in humans at 90 min after the ingestion of >375 mg of EGCG reached plateau level (15).

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Fig. 1. Dose-dependent reduction of mutation frequency by EGCG in the rpsL gene in the lung. Seven-week-old male mice were fed with EGCG in drinking water. One week later, they were given B[a]P by a single i.p. injection (500 mg/kg body wt). Two weeks after the B[a]P treatment, the mutation frequency in the rpsL gene was determined. Each column represents the mean ± SD. Statistical significance was determined using ANOVA. *Significant difference between the water and B[a]P treated groups (P < 0.05).
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Mice consumed ~3.5 ml/day of drinking water in this study. Average body weight of mice was ~25 g. The daily intake of EGCG was calculated to be ~7 mg/kg when mice were given 0.005% EGCG. This amount of EGCG corresponds to ~420 mg intake of EGCG in humans, assuming a body weight of 60 kg. In general, green tea contains 200700 mg/l EGCG (16,17). EGCG given to mice at 0.005% in drinking water corresponds to ~6002100 ml of green tea per day in humans. Tea-lovers take >10 cups of green tea. Indeed, a prospective cohort study of a Japanese population that included 8497 individuals revealed that 17.9% of the males and 13.1% of females consumed >10 cups of green tea per day. Approximately 70% of the subjects used medium-size (180 ml) teacups (16). They consumed >1800 ml of green tea per day. Thus, the amount of EGCG consumption almost corresponds to that of the mice in our study.
To investigate the effect of EGCG on the mutational spectrum, we analyzed the sequence of the rpsL gene. The summary of mutations found in the rpsL gene of the lung is shown in Table I
. The mutational spectrum induced by B[a]P was different from that observed in the lung from corn oil-treated mice, where a large proportion of the mutations were the A:T
C:G transversion. Treatment of mice with B[a]P caused an increase in G:C
T:A and G:C
C:G transversions. Thus, the relative ratio of A:T
C:G transversions decreased. The mutation frequency of each type of mutation in total mutations, and the relative ratio of each mutation, are summarized in Figure 2
. The frequency of G:C
A:T transitions, G:C
C:G and G:C
T:A transversions, one-base deletions and one-base insertions in the rpsL gene of B[a]P-treated mice were significantly higher than those seen in corn oil-treated mice. Approximately 67% (8/12) of one-base deletions occurred at the G:C base-pair in B[a]P-treated mice, whereas all of the one-base deletions occurred at the A:T base-pair in control mice. Deletion of the G:C base-pairs occurred in several poly(dG) sequences. These results show that B[a]P induced mutations mainly at the G:C base-pair. B[a]P is metabolically activated to BPDE, which is the ultimate carcinogenic form of B[a]P (18). BPDE binds to DNA and forms an adduct predominantly at the 2-amino group of guanine (18,19). To clarify hotspots of B[a]P-induced mutations in the rpsL gene, we analyzed the adjacent sequence pattern of the mutated G:C base-pairs (Figure 3
). The central guanine base in some nucleotide sequences such as AGG, CGG, CGT, TGG, TGC and GGT appeared to be the target of BPDE. The occurrence of the mutations was suppressed by the treatment of mice with 0.005% EGCG. The sequences in the hotspots between reported mutations in several oncogenes/tumor suppressor genes and B[a]P-induced mutations in the rpsL gene were compared. The results indicated that B[a]P-induced mutations in the rpsL gene had some motifs similar to mutational hotspots of lung cancer, namely TGG and GGT for the 12th codon of the Ki-ras gene, CGT for codons 157 and 273 of human p53 gene and CGG for codon 248 of human p53 gene. The activation of the Ki-ras gene and the inactivation of the p53 gene by mutations are common genetic lesions found in both human and rodent lung tumors. Ki-ras mutation occurred in 3050% of all the non-small cell lung cancers in humans (3,20,21). Mutations that occurred at guanine in the first or second base of codon 12 (GGT) were frequently observed in lung tumors in humans (22,23). Most of the pulmonary tumors in mice induced by B[a]P had mutations in the first and second base of the 12th codon of the Ki-ras gene (11GCT12GGT13GGC) (24,25). Approximately 6070% of human lung cancers have been reported to contain mutations in the p53 gene (21,26). There are several mutational hotspots in the p53 gene in the lung cancer gene in humans, such as those in codons 157, 248 and 273 (27). Strong selectivity in BPDE binding at guanine positions in codons 157 (156CGC157GTC158CGC), 248 (247AAC248CGG-249AGG) and 273 (272GTG273CGT274GTT) of human p53 gene was observed in HeLa cells treated with BPDE (27). These results suggest that mutations found in the rpsL gene may reflect the tumor-initiating potency of B[a]P in the lung, and that the intake of EGCG may reduce the mutation frequency that occurred in the Ki-ras gene or the p53 gene, leading to the reduction of tumors in the lung.

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Fig. 2. Frequencies of each mutation found in the rpsL gene of the lung from rpsL transgenic mice. (a) Corn oil-treated group; (b) B[a]P-treated group. Each column represents the mean ± SD. FS 1, one base deletion; FS + 1, one base insertion.
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In conclusion, this study clearly demonstrated the antimutagenic effects of EGCG for the B[a]P-induced mutagenesis in vivo, and suggests that drinking green tea might reduce the tumor-initiating potency of B[a]P in the lung.
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Acknowledgments
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This work was supported in part by a grant-in-aid from the Ministry of Education, Science, Sports and Culture of Japan, the Promotion of Fundamental Studies in Health Sciences of the Organization for Pharmaceutical Safety and Research (OPSR) of Japan and by research funds from the Japanese Society of Alternatives to Animal Experiments.
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Notes
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4 To whom correspondence should be addressed Email: kamataki{at}pharm.hokudai.ac.jp 
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References
|
---|
-
McCann,J., Choi,E., Yamasaki,E. and Ames,B.N. (1975) Detection of carcinogens as mutagens in the Salmonella/microsome test: assay of 300 chemicals. Proc. Natl Acad. Sci. USA, 72, 51355139.[Abstract]
-
Heidelberger,C. (1975) Chemical carcinogenesis. Annu. Rev. Biochem., 44, 79121.[ISI][Medline]
-
Straus,D.S. (1981) Somatic mutation, cellular differentiation and cancer causation [editorial]. J. Natl Cancer Inst., 67, 233241.[ISI][Medline]
-
Ames,B.N. (1979) Identifying environmental chemicals causing mutations and cancer. Science, 204, 587593.[ISI][Medline]
-
Xu,Y., Ho,C.T., Amin,S.G., Han,C. and Chung,F.L. (1992) Inhibition of tobacco-specific nitrosamine-induced lung tumorigenesis in A/J mice by green tea and its major polyphenol as antioxidants. Cancer Res., 52, 38753879.[Abstract]
-
Katiyar,S.K., Agarwal,R. and Mukhtar,H. (1993) Protective effects of green tea polyphenols administered by oral intubation against chemical carcinogen-induced forestomach and pulmonary neoplasia in A/J mice. Cancer Lett., 73, 167172.[ISI][Medline]
-
Katiyar,S.K., Agarwal,R., Zaim,M.T. and Mukhtar,H. (1993) Protection against N-nitrosodiethylamine and benzo[a]pyrene-induced forestomach and lung tumorigenesis in A/J mice by green tea. Carcinogenesis, 14, 849855.[Abstract]
-
Cao,J., Xu,Y., Chen,J. and Klaunig,J.E. (1996) Chemopreventive effects of green and black tea on pulmonary and hepatic carcinogenesis. Fund. Appl. Toxicol., 29, 244250.[ISI][Medline]
-
Wang,Z.Y., Wang,L.D., Lee,M.J., Ho,C.T., Huang,M.T., Conney,A.H. and Yang,C.S. (1995) Inhibition of N-nitrosomethylbenzylamine-induced esophageal tumorigenesis in rats by green and black tea. Carcinogenesis, 16, 21432148.[Abstract]
-
Yamane,T., Hagiwara,N., Tateishi,M. et al. (1991) Inhibition of azoxymethane-induced colon carcinogenesis in rat by green tea polyphenol fraction. Jpn. J. Cancer Res., 82, 13361339.[ISI][Medline]
-
Wang,Z.Y., Cheng,S.J., Zhou,Z.C., Athar,M., Khan,W.A., Bickers,D.R. and Mukhtar,H. (1989) Antimutagenic activity of green tea polyphenols. Mutat. Res., 223, 273285.[ISI][Medline]
-
Okuda,T., Mori,K. and Hayatsu,H. (1984) Inhibitory effect of tannins on direct-acting mutagens. Chem. Pharm. Bull., 32, 37553758.[ISI][Medline]
-
Gondo,Y., Shioyama,Y., Nakao,K. and Katsuki,M. (1996) A novel positive detection system of in vivo mutations in rpsL (strA) transgenic mice. Mutat. Res., 360, 114.[ISI][Medline]
-
Sambrook,J., Fritsh,E.F. and Maniatis,T. (1989) Molecular Cloning: A Laboratory Manual, 2nd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
-
Nakagawa,K., Okuda,S. and Miyazawa,T. (1997) Dose-dependent incorporation of tea catechins, ()-epigallocatechin-3-gallate and ()-epigallocatechin, into human plasma. Biosci. Biotech. Biochem., 61, 19811985.[ISI][Medline]
-
Imai,K., Suga,K. and Nakachi,K. (1997) Cancer-preventive effects of drinking green tea among a Japanese population. Prev. Med., 26, 769775.[ISI][Medline]
-
Graham,H.N. (1992) Green tea composition, consumption and polyphenol chemistry. Prev. Med., 21, 334350.[ISI][Medline]
-
Phillips,D.H. (1983) Fifty years of benzo[a]pyrene. Nature, 303, 468472.[ISI][Medline]
-
Meehan,T., Straub,K. and Calvin,M. (1977) Benzo[a]pyrene diol epoxide covalently binds to deoxyguanosine and deoxyadenosine in DNA. Nature, 269, 725727.[ISI][Medline]
-
Bos,J.L. (1989) ras oncogenes in human cancer: a review. Cancer Res., 49, 46824689.[Abstract]
-
Gao,H.G., Chen,J.K., Stewart,J., Song,B., Rayappa,C., Whong,W.Z. and Ong,T. (1997) Distribution of p53 and K-ras mutations in human lung cancer tissues. Carcinogenesis, 18, 473478.[Abstract]
-
Nakano,H., Yamamoto,F., Neville,C., Evans,D., Mizuno,T. and Perucho,M. (1984) Isolation of transforming sequences of two human lung carcinomas: structural and functional analysis of the activated c-K-ras oncogenes. Proc. Natl Acad. Sci. USA, 81, 7175.[Abstract]
-
Rodenhuis,S. and Slebos,R.J. (1992) Clinical significance of ras oncogene activation in human lung cancer. Cancer Res., 52, 2665s2669s.[Abstract]
-
Mass,M.J., Jeffers,A.J., Ross,J.A., Nelson,G., Galati,A.J., Stoner,G.D. and Nesnow,S. (1993) Ki-ras oncogene mutations in tumors and DNA adducts formed by benz[j]aceanthrylene and benzo[a]pyrene in the lungs of strain A/J mice. Mol. Carcinogen., 8, 186192.[ISI][Medline]
-
You,M., Candrian,U., Maronpot,R.R., Stoner,G.D. and Anderson,M.W. (1989) Activation of the Ki-ras protooncogene in spontaneously occurring and chemically induced lung tumors of the strain A mouse. Proc. Natl Acad. Sci. USA, 86, 30703074.[Abstract]
-
Hollstein,M., Sidransky,D., Vogelstein,B. and Harris,C.C. (1991) p53 mutations in human cancers. Science, 253, 4953.[ISI][Medline]
-
Denissenko,M.F., Pao,A., Tang,M. and Pfeifer,G.P. (1996) Preferential formation of benzo[a]pyrene adducts at lung cancer mutational hotspots in p53. Science, 274, 430432.[Abstract/Free Full Text]
Received July 6, 1998;
revised September 22, 1998;
accepted October 9, 1998.