Report on the mutagenicity of flavone derivatives and their contribution to advancing scientific knowledge. (2025)

Cancers are now understood to be due to the accumulation of genetic alterations in somatic cells, with numerous environmental factors playing a role in human carcinogenesis. In 1975, Ames, B.N. (University of California) and colleagues reported a highly valuable mutation test using Salmonella typhimurium to detect environmental carcinogens. (1) Many typical carcinogens have been found to exhibit mutagenic properties in the Salmonella test, leading to this method being widely adopted in research laboratories, industry, and regulatory agencies. (2-4) These findings have prompted the search for carcinogenic compounds as mutagens in the environment, particularly in human foods. A variety of flavonoids, including quercetin, kaempferol, and their glycosides rutin and astragalin, are commonly found in vegetables and fruits. Interestingly, bracken ferns, consumed by residents in certain countries such as Japan and Korea, are known to contain these flavonoids. (5,6) Notably, cows grazing on bracken fern-containing fields exhibited a high incidence of hematuria with urinary bladder tumors. (7) Additionally, rats fed on diets containing bracken fern developed urinary bladder tumors and intestinal adenocarcinomas. (8,9) However, during the 1970s, the specific carcinogenic components in bracken fern had not yet been fully identified.

Based on these observations, Sugimura, T. (National Cancer Center Research Institute, Tokyo) and colleagues conducted an investigation into the mutagenic properties of 11 flavone derivatives, including quercetin and kaempferol, using S. typhimurium TA98 and TA100. Their research yielded significant results, revealing that quercetin, kaempferol, and galangin exhibited notably high mutagenic activities in S. typhimurium TA98 with S9 mix. The mutagenic activity of these flavone derivatives was in the same order as that of the well-known mutagenic carcinogen benzo[a]pyrene. The presence of a 3,5,7-trihydroxyflavone structure appeared to be crucial for the exhibition of mutagenic activity by flavone derivatives. Fisetin, a 3,7-dihydroxy derivative, exhibited weak mutagenicity, whereas the remaining seven flavone derivatives showed no mutagenic activity at all. These novel and informative findings were published in Proc. Jpn. Acad. Ser. B in 1977. (10) The mutagenicity of quercetin and related compounds in S. typhimurium strains has been independently reported by other research groups. (11,12)

Furthermore, it was demonstrated that quercetin exerts mutagenic effects on a mouse lymphoma L5178 [TK.sup.+/-] mutation assay system and on Chinese hamster lung cells with diphtheria toxin resistance as a selective marker. (13,14) In contrast, no increase in HGPRT-deficient mutants on V79 Chinese hamster cells by quercetin was reported. (15) The genotoxic effect of quercetin in a micronucleus test was shown in mice receiving intraperitoneal injections. (16) However, there have been no other reports on in vivo genotoxic activity of quercetin in mice and rats by oral administration. (17,18) Thus, conflicting results concerning the genotoxicity of quercetin were reported in in vitro cultured mammalian cell tests and in vivo genotoxicity tests in animals. The reasons of these differences may be due to several factors, including the mammalian cell test system, dose, route of administration of quercetin, and the species and strain of animals tested.

The amounts of flavones and their glycosides are notably high in certain foods, particularly vegetables and fruits, with an estimated daily intake exceeding 100 mg per person. If flavones and their glycosides were indeed carcinogenic, humans would have been continuously and unavoidably exposed to plant-derived carcinogens in our daily lives. Therefore, the Ministry of Health and Welfare of Japan organized a research group for long-term animal carcinogenesis experiments involving flavonoids. As part of this program, feeding experiments of quercetin and its glycoside, rutin, were conducted using ACI rats, ddY mice, and golden hamsters. The chemical structures of quercetin and rutin are shown in Fig. 1. The outcomes of these three animal experiments revealed in 1980-1982 that flavonoids, such as quercetin and rutin, did not exhibit carcinogenic properties when included in diets at levels ranging from 1-10%. (19-21) Conversely, in 1980, a joint research group from the USA and Turkey reported that feeding quercetin to Norwegian rats led to the development of carcinomas in the urinary bladder and small intestine, even at a dietary concentration of 0.1%. (22) The reason for this discrepancy between the results obtained by the Japanese and the USA-Turkey groups remained unknown. There are several factors that may influence the carcinogenicity of quercetin. These include differences in the species and strains of the animals tested, the composition of the basal diet, and the production of endogenous carcinogens.

Consequently, the National Toxicology Program in the USA decided to include quercetin in studies conducted in rats of the F344 strain of both sexes. Ultimately, the outcome observed was the development of benign adenomas in the kidneys of males, a phenomenon linked to sex-dependent [alpha][2.sub.u]-globulin and unrelated to human carcinogenesis as reported in 1992.23) Collectively, these observations indicate that flavones, including quercetin and kaempferol and their glycosides, are presently not regarded as risk factors for human carcinogenesis. Moreover, the carcinogenic principal in bracken fern was identified and named ptaquiloside and aquilide A, which is a terpene compound not a flavonoid compound, by Japanese and Dutch scientists, respectively, in 1983-84. (24,25)

The issues surrounding flavonoids provided a warning regarding the simplistic understanding that mutagens are necessarily carcinogens. These concerns have raised questions regarding the reliability of using microbial tests to assess the safety of environmental compounds and new industrial chemicals. In fact, extensive genotoxicity assessments, involving a variety of chemicals have demonstrated that relying solely on microbial tests, including Ames' Salmonella test, is insufficient for safety assessment. As a result, a battery of tests for genotoxicity, including the in vitro Ames' Salmonella test, in vitro chromosomal aberration test, and in vivo micronucleus test, have been established and are now recommended for evaluating the safety of environmental compounds and new industrial chemicals. (26,27) Microbial tests, such as the Ames' test, are highly valuable for initial assessments of a substance's potential carcinogenicity. However, it is important to note that comprehensive, long-term animal tests are essential to confirm the safety of chemicals. These tests typically require an experimental period of approximately two years and are financially demanding. Therefore, alternative approaches involving animal tests that focus on precancerous lesions with relatively short experimental periods and using transgenic and knockout animals with increased susceptibility have been proposed and adopted.

The mechanism underlying quercetin, a compound widely distributed in the environment, exhibits mutagenicity in the Ames' test but does not induce cancer in animal experiments is yet to be fully elucidated. Several papers have demonstrated the chemopreventive potential of quercetin. For example, quercetin suppressed oral and lung carcinogenesis induced by chemical carcinogens. (28,29) A similar situation exists with other flavonoids. Quercetin has antioxidant and anti-inflammatory activity. (30,31) These biological properties of quercetin may have an overall impact on its in vivo carcinogenic activity in animals. These plant flavonoid issues serve as a reminder that genotoxicity tests utilizing Salmonella strains are not infallible, and their results must be considered with caution. Consequently, the informative report on the mutagenicity of flavone derivatives published in Proc. Jpn. Acad. Ser. B in 1977 by Sugimura, T. et al., (10) has made significant contributions to initiating and promoting genotoxicity studies of flavonoids and is now internationally highly evaluated.

doi: 10.2183/pjab.100.032

Acknowledgments

This study received support from Grants-in-aid for Research on the Risk of Chemical Substances from the Ministry of Health, Labour, and Welfare of Japan (JP22KD0101), and Research on Global Health Issues (US-Japan Cooperative Medical Sciences Program) from the Japan Agency for Medical Research and Development (JP23jk0210009).

References

(1) Ames, B.N., McCann, J. and Yamasaki, E. (1975) Methods for detecting carcinogens and mutagens with the Salmonella/mammalianmicrosome mutagenicity test. Mutat. Res. 31, 347-364.

(2) 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. U.S.A. 72, 5135-5139.

(3) Purchase, I.F., Longstaff, E., Ashby, J., Styles, J.A., Anderson, D., Lefevre, P.A. et al. (1976) Evaluation of six short term tests for detecting organic chemical carcinogens and recommendations for their use. Nature 264, 624-627.

(4) Sugimura, T., Sato, S., Nagao, M., Yahagi, T., Matsushima, T., Seino, Y. et al. (1976) Overlapping of carcinogens and mutagens. In Fundamentals in Cancer Prevention (eds. Magee, P.N., Takayama, S., Sugimura, T. and Matsushima, T). Japan Scientific Societies Press, Tokyo, pp. 191-215.

(5) Nakabayashi, T. (1955) Isolation of astragalin and isoquercitrin from bracken, Pteridium aquilinum. Bull. Agric. Chem. Soc. Jpn. 19, 104-109.

(6) Wang, C.Y., Pamukcu, A.M. and Bryan, G.T. (1973) Isolation of fumaric acid, succinic acid, astragalin isoquercitrin and tiliroside from Pteridium aquilinum. Phytochemistry 12, 2298-2299.

(7) Pamukcu, A.M. and Bryan, G.T. (1979) Bracken fern, a natural urinary bladder and intestinal carcinogen. In Naturally Occurring Carcinogens-Mutagens and Modulators of Carcinogenesis. (eds. Miller, E.C., Miller, J.A., Hirono, I., Sugimura, T. and Takayama, S.). Japan Scientific Societies Press, Tokyo; University Park Press, Baltimore, MD, pp. 89-99.

(8) Evans, I.A. and Mason, J. (1965) Carcinogenic activity of bracken. Nature 208, 913-914.

(9) Hirono, I., Shibuya, C., Fushimi, K. and Haga, M. (1970) Studies on carcinogenic properties of bracken, Pteridium aquilinum. J. Natl. Cancer Inst. 45, 179-188.

(10) Sugimura, T., Nagao, M., Matsushima, T., Yahagi, T., Seino, Y., Shirai, A. et al. (1977) Mutagenicity of flavone derivatives. Proc. Jpn. Acad. 23) Ser. B 53, 194-197.

(11) Bjeldanes, L.F. and Chang, G.W. (1977) Mutagenic activity of quercetin and related compounds. Science 197, 577-578.

(12) MacGregor, J.T. and Jurd, L. (1978) Mutagenicity of plant flavonoids: structural requirements for mutagenic activity in Salmonella typhimurium. Mutat. Res. 54, 297-309.

(13) Meltz, M.L. and MacGregor, J.T. (1981) Activity of the plant flavanol quercetin in the mouse lymphoma L5178Y [TK.sup.+/-] mutation, DNA single-strand break, and Balb/c 3T3 chemical transformation assays. Mutat. Res. 88, 317-324.

(14) Nakayasu, M., Sakamoto, H., Terada, M., Nagao, M. and Sugimura, T. (1986) Mutagenicity of quercetin in Chinese hamster lung cells in culture. Mutat. Res. 174, 79-83.

(15) van der Hoeven, J.C., Bruggeman, I.M. and De bets, F.M. (1984) Genotoxicity of quercetin in cultured mammalian cells. Mutat. Res. 136, 9-21.

(16) Sahu, R.K., Basu, R. and Sharma, A. (1981) Genetic toxicological testing of some plant flavonoids by the micronucleus test. Mutat. Res. 89, 69-74.

(17) MacGregor, J.T., Wehr, C.M., Manners, G.D., Jurd, L., Minkler, J.L. and Carrano, A.V. (1983) In vivo exposure to plant flavonols. Influence on frequencies of micronuclei in mouse erythrocytes and sister-chromatid exchange in rabbit lymphocytes. Mutat. Res. 124, 255-270.

(18) Utesch, D., Feige, K., Dasenbrock, J., Broschard, T.H., Harwood, M., Danielewska-Nikiel, B. et al. (2008) Evaluation of the potential in vivo genotoxicity of quercetin. Mutat. Res. 654, 38-44.

(19) Saito, D., Shirai, A., Matsushima, T., Sugimura, T. and Hirono, I. (1980) Test of carcinogenicity of quercetin, a widely distributed mutagen in food. Teratog. Carcinog. Mutagen. 1, 213-221.

(20) Hirono, I., Ueno, I., Hosaka, S., Takanashi, H., Matsushima, T., Sugimura, T. et al. (1981) Carcinogenicity examination of quercetin and rutin in ACI rats. Cancer Lett. 13, 15-21.

(21) Morino, K., Matsukara, N., Kawachi, T., Ohgaki, H., Sugimura, T. and Hirono, I. (1982) Carcinogenicity test of quercetin and rutin in golden hamsters by oral administration. Carcinogenesis 3, 93-97.

(22) Pamukcu, A.M., Yalciner, S., Hatcher, J.F. and Bryan, G.T. (1980) Quercetin, a rat intestinal and bladder carcinogen present in bracken fern (Pteridium aquilinum). Cancer Res. 40, 3468-3472.

(23) U.S. Department of Health and Human Services. (1992) NTP Technical Report (No. 409) on the Toxicology and Carcinogenesis Studies of Quercetin (CAS No. 117-39-5) in F344/N Rats (Feed Studies). U.S. Department of Health and Human Services, Public Health Service, National Institute of Health, Research Triangle Park, NC.

(24) Hirono, I., Yamada, K., Niwa, H., Shizuri, Y., Ojika, M., Hosaka, S. et al. (1984) Separation of carcinogenic fraction of bracken fern. Cancer Lett. 21, 239-246.

(25) van der Hoeven, J.C., Lagerweij, W.J., Posthu mus, M.A., van Veldhuizen, A. and Holterman, H.A. (1983) Aquilide A, a new mutagenic compound isolated from bracken fern (Pteridium aquilinum (L.) Kuhn). Carcinogenesis 4, 1587-1590.

(26) European Food Safety Authority (EFSA), An dreoli, C., Aquilina, G., Bignami, M., Bolognesi, C., Crebelli, R. et al. (2023) Harmonised approach for reporting reliability and relevance of genotoxicity studies. EFSA Support. Publ. 20, EN-8270.

(27) ICH Harmonised Tripartite Guideline. (2011) Guidance on genotoxicity testing and data interpretation for pharmaceuticals intended for human use S2 (R1). Current Step 4 Version, dated 9 November 2011.

(28) Makita, H., Tanaka, T., Fujitsuka, H., Tatematsu, N., Satoh, K., Hara, A. et al. (1996) Chemoprevention of 4-nitroquinoline 1-oxide-induced rat oral carcinogenesis by the dietary flavonoids chalcone, 2-hydroxychalcone, and quercetin. Cancer Res. 56, 4904-4909.

(29) Khanduja, K.L., Gandhi, R.K., Pathania, V. and Syal, N. (1999) Prevention of Nnitrosodiethylamine-induced lung tumorigenesis by ellagic acid and quercetin in mice. Food Chem. Toxicol. 37, 313-318.

(30) Middleton, E., Jr., Kandaswami, C. and Theo harides, T.C. (2000) The effects of plant flavonoids on mammalian cells: implications for inflammation, heart disease, and cancer. Pharmacol. Rev. 52, 673-751.

(31) Erlund, I. (2004) Review of the flavonoids quercetin, hesperetin, and naringenin. Dietary sources, bioactivities, bioavailability, and epidemiology. Nutr. Res. 24, 851-874.

(Received Nov. 30, 2023; accepted Mar. 27, 2024)

43. Mutagenicity of Flavone Derivatives (*)

By Takashi SUGIMURA, (**), (***) Minako NAGAO, (**) Taijiro MATSUSHIMA, (***) Takie YAHAGI, (**) Yuko SEINO, (**) Atsuko SHIRAI, (***) Mutsuko SAWAMURA, (***) Shinsaku NATORI, (****) Kunitoshi YOSHIHIRA, (****) Masamichi FUKUOKA, (****) and Masanori KUROYANAGI (****)

(Communicated by Hamao Umezawa, M.J.A., Sept. 12, 1977)

Bracken has long been considered to contain carcinogenic principles (Evans and Mason, 1965; Hirono et al., 1970), however, isolation of carcinogenic compounds from bracken has not been successful. Overlapping of mutagens and carcinogens is now well established (McCann et al., 1975; Sugimura et al., 1976). We found that a flavonoid compound, kaempferol isolated from bracken was mutagenic. This finding prompted us to investigate mutagenicities of other flavone compounds, which were known to exist in various plants (Harborne et al., 1975; Harborne, 1977).

Kaempferol was extracted and purified from bracken. Quercetin and fisetin were purchased from Tokyo Kasei Kogyo Co., galangin from Fulka AG., chrysin and flavone from Aldrich Chemical Co., and 3-hydroxyflavone from Eastman Kodak Co. Isoflavone derivatives, daizein, genistein and orobol were kindly provided by Dr. Tomio Takeuchi, Institute of Microbial Chemistry, Tokyo. Naringenin, a flavanone derivative, was purchased from Tokyo Kasei Kogyo Co. The purities of these compounds were confirmed by thin-layer chromatography.

Salmonella typhimurium TA98 and TA100 were used (Ames et al., 1975). Test compounds were preincubated with S-9 Mix from the liver of rats which had been treated by polychlorinated biphenyl as described previously (Nagao et al., 1977).

Table I shows the structures of the substances tested and their mutagenic activities in terms of number of revertants per nmole. Kaempferol, quercetin and galangin showed significantly high mutagenic activities. It is suggested from their structures that the hydroxy groups on their phenyl moiety would not be essential in the mutagenic activity, but the 3,5,7-trihydroxyflavone structure would be important in exhibition of the mutagenic action. Fisetin which was a 3,7-dihydroxy derivative was weakly mutagenic, but chrysin, a 5,7-dihydroxy compound, did not show any mutagenic activity. 3-Hydroxyflavone and flavone were non-mutagenic. None of the three isoflavones tested was mutagenic at all. Flavanone (not listed in Table I) and 5,7,4'-trihydroxyflavanone, named naringenin, which is known to be present in fruits such as grapefruit (Dunlap and Wender, 1962) were not mutagenic at all.

Many flavonoids exist as glycosides in plants. A typical example is rutin, which is the 3-rutinoside of quercetin. Astragalin which was found in bracken is the 3-glucoside of kaempferol (Kuroyanagi et al., 1974; Nakabayashi, 1955). Extensive studies on the mutagenicities of various flavones and their glycosides are now in progress in our laboratories.

The mutagenicities of kaempferol and quercetin are in the same order as those of o-aminoazotoluene and 4-aminobiphenyl with TA98 and in the same order as that of 3/-methyl-4-dimethylaminoazobenzene with TA100, under the standardized conditions used in our laboratories. Although quercetin and several other flavone compounds have been reported to be non-carcinogenic (DeEds, 1968), the results described here indicate the need for further carcinogenic tests on flavone derivatives. Long-term in vivo experiments are now in progress on the carcinogenicities of kaempferol, quercetin and rutin. Carcinogens in natural foods should be more and more studied as well as those in cooked foods, cigarette smoke, polluted air and water, etc.

(*) This work was supported in part by Grant-in-Aid for Cancer Research from the Ministry of Education, Culture and Science, and the Ministry of Health and Welfare.

(**) National Cancer Center Research Institute, Tsukiji, Chuo-ku, Tokyo 104.

(***) Institute of Medical Science, University of Tokyo, Shirokanedai, Minatoku, Tokyo 108.

(****) National Institute of Hygienic Sciences, Yoga, Setagaya-ku, Tokyo 158.

References

Ames, B. N., McCann, J., and Yamasaki, E. (1975) : Mutation Res., 31, 347-364.

DeEds, F. (1968) : Comprehensive Biochem., 20, 127-171.

Dunlap, W. J., and Wender, S. H. (1962) : Anal. Biochem., 4, 110-115.

Evans, I. A., and Mason, J. (1965) : Nature, 208, 913-914.

Harborne, J. B. (1977) : Biochem. System. Ecol., 5, 7-22.

Harborne, J. B., Mabry, T. J., and Mabry, H. (1975) : The Flavonoids. Chapman & Hall, London.

Hirono, I., Shibuya, C., Fushimi, K., and Haga, M. (1970) : J. Natl. Cancer Inst., 45, 179-188.

Kuroyanagi, M., Fukuoka, M., Yoshihira, K., and Natori, S. (1974) : Chem. Pharm. Bull. Tokyo, 22, 2762-2764.

McCann, J., Choi, E., Yamasaki, E., and Ames, B. N. (1975) : Proc. Natl. Acad. Sci. US., 72, 5135-5139.

Nakabayashi, T. (1955) : Bull. Agr. Chem. Soc. Japan, 19, 104-109.

Nagao, M., Yahagi, T., Seino, Y., Sugimura, T., and Ito, N. (1977) : Mutation Res., 42, 385-342.

Sugimura, T., Sato, S., Nagao, M., Yahagi, T., Matsushima, T., Seino, Y., Takeuchi, M., and Kawachi, T. (1976) : Fundamentals in Cancer Prevention (eds, P. N. Magee, et al.), 191-215. Univ. of Tokyo Press, Tokyo.

By Yukari Totsuka [*1] [ID], and Keiji Wakabayashi [*2], ([dagger]) [ID]

(Edited by Takao SEKIYA, M.J.A.)

[*1] Department of Environmental Health Sciences, Hoshi University, Tokyo, Japan.

[*2] Graduate Division of Nutritional and Environmental Sciences, University of Shizuoka, Shizuoka, Japan.

([dagger]) Correspondence should be addressed to: K.

Wakabayashi, Graduate Division of Nutritional and Environmental Sciences, University of Shizuoka, 52-1 Yada, Surugaku, Shizuoka 422-8526, Japan (e-mail: kwakabayashi@ u-shizuoka-ken.ac.jp).

This paper commemorates the 100th anniversary of this journal and introduces the following paper previously published in this journal. Sugimura, T., Nagao, M., Matsushima, T., Yahagi, T., Seino, Y., Shirai, A., Sawamura, M., Natori, S., Yoshihira, K., Fukuoka, M. and Kuroyanagi, M. (1977) Mutagenicity of flavone derivatives. Proc. Jpn. Acad. Ser. B 53 (4), 194-197 (https://doi.org/10.2183/pjab.53.194).

Caption: Fig. 1. Structures of quercetin and rutin.

Table I. The structures and mutagenic activities offlavone derivativesName StructureFlavone Kaempferol [Please download the PDF to view the formula]Quercetin [Please download the PDF to view the formula]Galangin [Please download the PDF to view the formula]Fisetin [Please download the PDF to view the formula]Chrysin [Please download the PDF to view the formula]3-Hydroxyflavone [Please download the PDF to view the formula]Flavone [Please download the PDF to view the formula]Isoflavone Daizein [Please download the PDF to view the formula]Genistein [Please download the PDF to view the formula]Orobol [Please download the PDF to view the formula]Flavanone [Please download the PDF to view the formula] NaringeninName [His.sup.+] revertants/nmole * TA98 TA100Flavone Kaempferol 3.9 0.68Quercetin 5.3 0.35Galangin 3.8 1.6Fisetin 0.12 0.35Chrysin 0 03-Hydroxyflavone 0 0Flavone 0 0Isoflavone Daizein 0 0Genistein 0 0Orobol 0 0Flavanone Naringenin 0 0* Using S-9 Mix.