Proceedings of the Japan Academy, Series B
Online ISSN : 1349-2896
Print ISSN : 0386-2208
ISSN-L : 0386-2208
Review Series to Celebrate Our 100th Volume
Report on the mutagenicity of flavone derivatives and their contribution to advancing scientific knowledge
Yukari TOTSUKAKeiji WAKABAYASHI
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2024 Volume 100 Issue 10 Pages 537-544

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Abstract

Flavonoids, such as quercetin and kaempferol, and their glycosides, are widely distributed in vegetables and fruits. Sugimura, T. et al. investigated the mutagenicity of flavone derivatives, and found that quercetin and kaempferol showed high mutagenic activities in Salmonella typhimurium TA98 with S9 mix, comparable to that of the typical carcinogen, benzo[a]pyrene. These novel findings were published in Proc. Jpn. Acad. Ser. B 53, 194-197, 1977. Other research groups also reported the mutagenic properties of flavone derivatives in S. typhimurium strains. These observations led to the commencement of long-term animal carcinogenesis experiments involving quercetin. A USA-Turkey joint study reported that feeding rats with 0.1% quercetin in the diet produced carcinomas. However, Japanese scientists showed no carcinogenicity with quercetin in rats, mice, or hamsters, even at 10% in the diet. NTP Technical Report on the Toxicology and Carcinogenesis Studies of Quercetin in F344/N Rats concluded that there was no evidence of its carcinogenic activity. Therefore, the potential risk of quercetin in human cancers is likely to be negligible. These flavonoid issues provided a warning regarding the simplistic understanding that mutagens are carcinogens, and microbial tests alone are inadequate for safety assessment; therefore, and a battery of tests for genotoxicity is recommended. Thus, the informative report in 1977 made significant contributions to initiating and promoting genotoxicity studies of flavonoids.

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+/- 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.

Fig. 1

Structures of quercetin and rutin.

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 α2u-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.

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).

Notes

Edited by Takao SEKIYA, M.J.A.

Correspondence should be addressed to: K.Wakabayashi, Graduate Division of Nutritional and Environmental Sciences, University of Shizuoka, 52-1 Yada, Suruga-ku, Shizuoka 422-8526, Japan (e-mail: kwakabayashi@u-shizuoka-ken.ac.jp).

Footnotes

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).

References
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Appendices

[From Proc. Jpn. Acad. Ser. B, Vol. 53 No. 4, pp. 194-197 (1977)]

 
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