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Unaltered Meiotic Chromosome Segregation in Drosophila melanogaster Raised on a 5% Quercetin Diet
Food and Chemical Toxicology 36 (1998) 585±589
Unaltered Meiotic Chromosome Segregation in Drosophila melanogaster Raised on a 5% Quercetin Diet 1
D. D. SCHRAMM*{1, H. E. COLLINS*2, R. S. HAWLEY2 and J. B. GERMAN1
Department of Food Science and Technology and 2Department of Genetics, Section of Molecular and Cellular Biology, University of California at Davis, Davis, CA 95616, USA (Accepted 1 November 1997)
AbstractÐFlavonoid plant pigments are an integral part of the human diet. Although potentially negative mitotic eects of ¯avonoids have been observed in model organisms, investigation into meiotic eects of ¯avonoids has been neglected. As ¯avonoids aect cell signalling and DNA replication, and because the ¯avonoid content of the human food supply is being increased, determining the eects of ¯avonoids on meiotic ®delity is important. Here, the eect of the human food supply's most prevalent ¯avonoid, quercetin, on the level of meiotic recombination and the amount of X and 4th chromosome non-disjunction in Drosophila melanogaster females was determined. This model organism was chosen since Drosophila melanogaster and Homo sapiens share a remarkable number of commonalties in the meiotic processes of oogenesis and because genetic techniques allow a detailed analysis of meiotic processes in Drosophila. No signi®cant eect on either non-disjunction levels or the percentage distribution of exchange bivalents was observed. A signi®cant eect was observed on the number of ospring; F1 and F2 generations of ¯ies raised on a quercetin diet produced over 10% more progeny than ¯ies raised on a control diet. In this investigation, high quercetin consumption by Drosophila melanogaster females did not pose a threat to meiotic ®delity. # 1998 Elsevier Science Ltd. All rights reserved Keywords: quercetin; ¯avonoid; reproduction; recombination; Drosophila melanogaster; meiosis.
INTRODUCTION
Flavonoids are secondary metabolites synthesized by plants and consumed by humans in fruits, vegetables, legumes, and plant-derived products such as coee, tea, wine and fermented foods (Middleton and Kandaswami, 1993). Human ¯avonoid consumption has been estimated to be in the range of 1 g/day (Kuhnau, 1976), and is increasing for a variety of reasons. First, crop engineers are pursuing increased plant ¯avonoid content with the intent of improving disease resistance in plants and plant nodulation eciency (Dixon et al., 1996; Henkel, 1995). Secondly, ¯avonoids are being considered as potential food additives since they can reduce food spoilage (Grange and Davey, 1990; Nelson, 1982; Ramanathan and Das, 1992; Zeiger, 1993). Lastly, *These authors and their laboratories contributed equally to this work. {Address for correspondence.
corporate marketing has persuaded people to purchase semi-pure ¯avonoids as supplements, using data demonstrating the ability of ¯avonoids to inhibit experimentally induced pathology in animal models (Cook and Samman, 1996; Odetti et al., 1990; Sakaguchi et al., 1992; Voskresenskii and Bobyrev, 1978). Although ¯avonoids inhibit pathology in a variety of experimental systems, potentially detrimental eects of ¯avonoids have been reported. Flavonoids can be mutagenic in the Ames Salmonella test, Drosophila melanogaster recessive sex-linked lethal test, and DNA ®ngerprinting cell culture tests (Bjeldans and Chong, 1977; Suzuki et al., 1991; Watson, 1982). They can disrupt the double helical con®guration of DNA, alter nucleic acid synthesis, and inhibit enzymes such as DNA and RNA polymerase and reverse transcriptase (RT) (Alvi et al., 1986; Austin et al., 1992; Formica and Regelson, 1995; Ono and Nakane, 1990). Furthermore, ¯avo-
0278-6915/98/$19.00+0.00 # 1998 Elsevier Science Ltd. All rights reserved. Printed in Great Britain PII S0278-6915(98)00013-1
586
D. D. Schramm et al.
noids can alter the synthesis and degradation of cell signalling molecules such as the cyclic nucleotides and the eicosanoids. As molecular mechanisms through which ¯avonoids could aect meiotic processes are known and the ¯avonoid content of the human diet is increasing, determining whether ¯avonoids alter meiosis in a detrimental manner is important. Meiotic non-disjunction in humans is the leading cause of human prenatal death and leads to pathology such as Down's syndrome (Sherman et al., 1994). Investigation of possible causes of meiotic non-disjunction in model organisms with similar meiotic processes is an important ®rst step towards understanding the causes in humans. In this experiment, Drosophila melanogaster females were used as model organisms since many aspects of their meiotic process are remarkably similar to those in humans (Koehler et al., 1996). Quercetin was used as a model ¯avonoid since it is the most prevalent ¯avonoid in the human diet and because several mechanisms through which quercetin aects biological processes are known. Females heterozygous for a variety of X chromosome markers were raised on a 5% quercetin diet and then crossed individually to appropriate tester males. The progeny of this cross were analysed to determine the fate of the maternal chromosomes in meiosis. This experimental design allowed identi®cation of eects of quercetin on the amount of progeny produced, the level of meiotic recombination, and the amount of X and 4th chromosome non-disjunction.
MATERIALS AND METHODS
Drosophila melanogaster stocks The X chromosome referred to as MF2 consists of the markers y sc cv v f car, as described previously (Koehler et al., 1996). All chromosome markers are fully recessive and, when homozygous, are scorable by phenotype. Wild-type stock, Oregon RM, was obtained from the Bloomington stock center (Bowling Green, OH, USA). MF2 was kept in stock as MF2/Y C(1)DX/Y, where C(1)DX is an attached chromosome (two X chromosomes physically fused together). The Y chromosome carried a dominant marker Bar of Stone (Bs) to allow identi®cation of XXY females. This experimental design ensured that only primary non-disjunctional events were included in the study (Koehler et al., 1996). Females heterozygous for the MF2 X chromosome and a wild-type X chromosome were crossed to tester males bearing an attached-XY compound chromosome. This attached-XY chromosome was marked with the dominant eye shaped mutation Bar (B) so it could be identi®ed in the presence of another X chromosome.
Food preparation Control food contained the following standard ingredients: 9 g agar (Moorhead and Co., Van Nuys, CA, USA), 40 g nutritional yeast (E.T. Horn, La Mirada, CA, USA), 90 g corn meal, 100 g glucose (Westco, Rancho Cordova, CA, USA), 1 litre water, 3 ml propionic acid (Fisher Scienti®c, Santa Clara, CA, USA), and 2 g n-butyl-hydroxybenzoate (Sigma, St Louis, MO, USA). Ingredients were mixed, boiled, and then cooled, poured into bottles, and allowed to harden. 5 mg yeast was added to the top of each bottle, 2 mg to each vial. Quercetin diets contained control food with 5% quercetin by weight. Quercetin was purchased from Sigma and used without further puri®cation. Quercetin was mixed thoroughly into the food immediately before pouring. The amount of food in control and experimental bottles was equal; food was aliquotted such that all parental ¯ies and progeny larva had access to excess food (i.e. 40 parental ¯ies/bottle; 50 ml food/bottle). The female progeny collected from these bottles were transferred to vials containing 8 ml of control food prepared exactly as for bottles. Experimental design Experimental design is diagrammed in Fig. 1. Virgin wild-type females were crossed to MF2 males and placed in control or 5% quercetin bottles on Day 0. On Day 7, the ¯ies were discarded. The progeny resulting from this mating were collected daily from Day 10 until Day 18. F1 progeny were counted and virgins heterozygous for the maternal chromosome markers were collected. These females, which developed on either control or quercetin diets, were placed individually in vials containing control food and mated to attached-XY tester males for 7 days. Progeny from this mating were counted 15 days after the parental ¯ies were ®rst placed into vials, and male ¯ies were scored for every MF2 marker. As recombination could occur in the mother, males could inherit all MF2 markers, none, or a recombinational product. Male ¯ies expressed all markers inherited from the maternal chromosome since the males contained only a maternal X chromosome. The protocol described allowed determination of the level of recombination in each maternal oocyte. Scoring and statistical analysis Chromosome markers were scored based on their phenotypes, as described previously (Lindsley and Zimm, 1992). Bottles and vials were scored individually, and males were scored separately from females. Unscorable ¯ies (due to broken wings, etc.) were discarded and not included in total counts. Tetrad analysis was calculated as previously described (Weinstein, 1936). Statistical analysis was conducted using Microsoft Excel's (Microsoft Corp., Atlanta, GA, USA) Single Factor Anova,
Meiotic segregation and quercetin
587
Fig. 1. Diagrammatic representation of experimental crosses.
testing the hypothesis that means from samples were equal. In addition, chi-square analysis was performed to determine if the expected (control) and experimental (quercetin) population distributions diered. RESULTS
Eects of quercetin on levels of non-disjunction and mitotic mutation Data on non-disjunction levels in ¯ies raised on control and 5% quercetin diets are presented in Table 1. ANOVA and chi-square analysis indicated no signi®cant dierences between control and quercetin groups. The percentage of non-disjunctional progeny in control ¯ies was 0.08%; the percentage in the progeny of ¯ies raised on the quercetin diet was 0.14%. Eects of quercetin on the percentage distribution of bivalents among the F2 generation The percentage of bivalents with zero (E0), one (E1), two (E2) and three (E3) crossovers is presented in Table 2. Oocytes from female ¯ies raised on a control diet had a tetrad distribution as previously reported (Koehler et al., 1996). Oocytes from females ¯ies raised on a 5% quercetin diet had a similar percentage of bivalents with zero, one or two crossovers. The percentage of E3 bivalents, however, was 0.9% in oocytes from ¯ies raised on the quercetin diet, compared with 0.0% in oocytes
from control ¯ies. This is noteworthy because the normal amount of E3 bivalents found in tetrad analysis of control ¯ies is virtually zero (Merriam and Frost, 1964). ANOVA and chi-square analysis indicated no signi®cant dierences between control and quercetin groups. Eects of quercetin on the number of ospring: MF2 males X wild-type females The eects of dietary quercetin on the number of ospring from Drosophila melanogaster are reported in Table 3. Parent (F0 generation) ¯ies placed on a 5% quercetin diet produced signi®cantly more ospring (F1 generation) than ¯ies placed on the control diet. In addition, female F1 ¯ies removed from bottles with quercetin food, placed on control food, and crossed to attached-XY males raised on control food produced signi®cantly more progeny (F2 generation) than the same cross with ¯ies from control bottles. Quercetin consumption increased male and female progeny equally. In addition, a noticeable but non-signi®cant delay in oocyte development occurred in progeny of ¯ies raised on 5% quercetin diets. Previously, quercetin was observed to induce delays in the development of oocytes from clam and star®sh (Eckberg, 1983). Table 2. Eect of quercetin on the % distribution of bivalents among the F2 generation Exchange ranka E0 E1 E2 E3
Table 1. Eect of quercetin on the amount of non-disjunctional progeny in the F2 generation
Control 5% Quercetin
Total ¯ies
Nondisjunctional progeny
Nondisjunction (%)
5102 5645
4 12
0.08 0.14
Chi-square analysis indicated no signi®cant dierences between control and quercetin groups (P = 0.068).
a
Flies raised on control diet 5.3% 72.4 22.2 0.0
Flies raised on the 5% quercetin diet 8.7%b 68c 22.4d 0.9e
Exchange rank indicates the fraction of chromosomes recovered in each case derived from bivalents with zero (Eo), one (E1), two (E2) or three (E3) exchanges. Tetrad analysis for regular progeny was calculated according to Weinstein (1936). Chi-square analysis indicated no signi®cant dierences between control and quercetin values. bP = 0.348. cP = 0.504. d P = 0.923. eP = 0.534.
588
D. D. Schramm et al. Table 3. Eect of quercetin on the number of Drosophila melanogaster ospring Number of ¯ies/container$
F1 Generation F2 Generation
Containers*
Control
5% Quercetin
% Control
P
5 243
10202 73 8.88 2 6.1
11292 59 10.472 6.3
110.7 117.9
0.03 0.05
*F1 generation ¯ies were raised in bottles and F2 generation in vials. $F1 numbers are the total number of ¯ies in each bottle over eight scoring days and F2 numbers are the number of male ¯ies per scoring day. Statistical analysis was conducted using ANOVA with signi®cance assigned at P < 0.05.
DISCUSSION
In this experiment, dietary quercetin did not signi®cantly alter the rate of meiotic non-disjunction in Drosophila melanogaster females. The percentage of non-disjunction in both control ¯ies and ¯ies raised on a quercetin diet was very similar to the frequency of 0.05% previously reported for similarly marked control ¯ies (Merriam and Frost, 1964). Quercetin's lack of eect on meiotic non-disjunction was unexpected since quercetin is known to inhibit the activity of kinase, polymerase and topoisomerase, disrupt the double helical con®guration of DNA, and alter the synthesis and degradation of cyclic nucleotides involved in the meiotic process (Havsteen, 1983; Middleton and Kandaswami, 1993). In addition to not eecting the amount of nondisjunction, the quercetin diet did not aect tetrad distribution. The trend towards an increase in ¯ies with an unusually large number of exchanges (eE3), from 0.0% in ¯ies on control diets to 0.9% in ¯ies on 5% quercetin diets, suggests the possibility of an eect on meiotic interference. Current theory proposes that the meiotic machinery required for exchange is limited (Loidl, 1994). Thus, if quercetin had increased the amount of meiotic machinery, increased recombination would have been expected; no increased recombination level was observed. In contrast, if interference were reduced, ¯ies with a large number of exchanges could occur with increased frequency without a signi®cant eect on the distribution of exchange. Flavonoids alter a variety of biochemical processes that can impact reproduction in plants and animals. In plants, ¯avonoids are used as sex hormones and can be essential for male gametophyte development (Birch et al., 1953; Burbulis et al., 1996; van der Meer et al., 1992). In insects, ¯avonoids can act as anti- or profeedants, and inhibit or stimulate growth (Hughes, 1988; McFarlane and Distler, 1982; Stermitz, 1981). Here, dietary quercetin increased the number of ospring produced by Drosophila melanogaster. Further investigation should determine whether this increase in ospring is due to increased fertility or increased survival rates. Although the number of ospring from Drosophila melanogaster was increased in this investigation, Stoewsand et al. (1984) noted no increased
progeny from rats raised on quercetin. In addition, oestrogenic ¯avonoids may reduce fertility in vertebrate herbivores (Hughes, 1988). In mammals, therefore, reproduction may not be stimulated by a high quercetin diet. Literature relating the eects of ¯avonoids on mitotic processes in vitro and in vivo is abundant. In contrast, investigation into meiotic eects of ¯avonoids has been neglected. Data from this investigation show only non-signi®cant eects of a 5% quercetin diet on the levels of meiotic recombination and the amount of X and 4th chromosome non-disjunction in ¯ies. This is important, since the meiotic processes of oogenesis in Drosophila melanogaster and Homo sapiens share a remarkable number of commonalties and because non-disjunction is the cause of several genetic diseases in humans including Down's syndrome (Koehler et al., 1996). In addition, although humans currently consume a diet that is less than 1% ¯avonoid by weight, human ¯avonoid consumption is increasing due to crop engineering, the addition of ¯avonoids to food as additives, and through the consumption of puri®ed compounds purchased from health food stores. If the eects of quercetin represent eects of dietary ¯avonoids as a whole and the human meiotic process is aected by quercetin as was that of Drosophila melanogaster, current or increased ¯avonoid intake by humans will not pose a risk to meiotic ®delity. AcknowledgementsÐThis investigation was supported in part by USDA/BARD grant IS2260-93RC. We thank Cora Morgan for editing, Je Sekelsky for technical advice, and Phyllis Wescott for food preparation. REFERENCES
Alvi N. K., Rizvi R. Y. and Hadi S. M. (1986) Interaction of quercetin with DNA. Bioscience Reports 6, 861±868. Austin C. A., Patel S., Ono K., Nakane H. and Fisher L. M. (1992) Site-speci®c DNA cleavage by mammalian DNA topoisomerase II induced by novel ¯avone and catechin derivatives. Biochemical Journal 282, 883±889. Birch A. J., Donovan F. W. and Moewus F. (1953) Biogenesis of ¯avonoids in Chlamydomonas eugametos. Nature 172, 902±904. Bjeldans L. F. and Chong G. W. (1977) Mutagenic activity of quercetin and related compounds. Science 197, 577±578. Burbulis I. E., Iacobucci M. and Shirley B. W. (1996) A null mutation in the ®rst enzyme of ¯avonoid biosyn-
Meiotic segregation and quercetin thesis does not aect male fertility in Arabidopsis. Plant Cell 8, 1013±1025. Cook N. C. and Samman S. (1996) FlavonoidsÐchemistry, metabolism, cardioprotective eects, and dietary sources. Nutritional Biochemistry 7, 66±76. Dixon R. A., Lamb C. J., Masoud S., Sewalt V. J. and Paiva N. L. (1996) Metabolic engineering: prospects for crop improvements through the genetic manipulation of phenylpropanoid biosynthesis and defense responsesÐa review. Gene 179, 61±71. Eckberg W. R. (1983) The eects of quercetin on meiosis initiation in clam and star®sh oocytes. Cell Dierentiation 12, 329±334. Formica J. V. and Regelson W. (1995) Review of the biology of quercetin and related bio¯avonoids. Food and Chemical Toxicology 33, 1061±1080. Grange J. M. and Davey R. W. (1990) Antibacterial properties of propolis. Journal of the Royal Society of Medicine 83, 159±160. Havsteen B. (1983) Flavonoids, a class of natural products of high pharmacological potency. Biochemical Pharmacology 32, 1141±1148. Henkel J. (1995) Genetic engineering: Fast forwarding to future foods. FDA Consumer 4, 6±11. Hughes C. L. (1988) Phytochemical mimicry of reproductive hormones ad modulation of herbivore fertility by phytoestrogens. Environmental Health Perspectives 78, 171±174. Knowles B. and Hawley R. S. (1991) Genetic analysis of microtubule motor proteins in Drosophila: A mutation at the NCD locus is a dominant enhancer of nod. Proceedings of the National Academy of Sciences of the U. S. A. 88, 7165. Koehler K., Boulton C., Collins H., French R., Herman K., Lace®eld S., Madden L., Schuetz C. and Hawley R. S. (1996) Spontaneous X chromosome MI and MII nondisjunction events in Drosophila melanogaster oocytes have dierent recombinational histories. Nature Genetics 14, 406±414. Kuhnau J. (1976) The ¯avonoids. A class of semi-essential food components: their role in human nutrition. World Review in Nutrition and Diet 24, 117±191. Lindsley D. L. and Zimm G. G. (1992) The Genome of Drosophila Melanogaster. Academic Press, San Diego. Loidl J. (1994) Cytological aspects of meiotic recombination. Birkhauser Verlag 50, 285±293. McFarlane J. E. and Distler M. H. W. (1982) The eect of rutin on growth fecundity and food utilization in Acheta domesticus. Journal of Insect Physiology 28, 85± 88. Merriam J. R. and Frost J. N. (1964) Examining the nondisjunction of the X chromosome in female Drosophila melanogaster. Genetics 49, 109±122. Middleton E. and Kandaswami C. (1993) The impact of plant ¯avonoids on mammalian biology: implications for immunity, in¯ammation, and cancer. In The
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Flavonoids. Edited by J. B. Harborne. pp. 619±662. Chapman and Hall, London. Nelson D. B. (1982) Letter (April 25) to F. R. Senti; reported in Life Sciences Research Oce Evaluation of the Health Aspects of Hesperidin, Naringin, and Citrus Bio¯avonoid Extracts as Food Ingredients, FASEB Journal 9. Odetti P. R., Borgoglio A., De Pascale A., Rolandi R. and Adezati L. (1990) Prevention of diabetes-increased aging eect on rat collagen-linked ¯uorescence by aminoguanidine and rutin. Diabetes 39, 796±801. Ono K. and Nakane H. (1990) Mechanisms of inhibition of various cellular DNA and RNA polymerases by several ¯avonoids. Journal of Biochemistry 108, 609±613. Ramanathan L. and Das N. P. (1992) Studies on the control of lipid oxidation in ground ®sh by some polyphenolic natural products. Journal of Agricultural and Food Chemistry 40, 17±21. Sakaguchi Y., Maehara Y., Baba H., Kusumoto T., Sugimachi K. and Newman R. A. (1992) Flavone acetic acid increases the antitumor eect of hyperthermia in mice. Cancer Research 52, 3306±3309. Sherman S. L., Peterson M. V., Freeman S. V., Hersey J., Pettay D., Taft L., Frantzen M., Mikkelsen M. and Hassold T. J. (1994) Non-disjunction of chromosome 21 in maternal meiosis I: evidence for a maternal agedependent mechanism involving reduced recombination. Human Molecular Genetics 3, 1365±1371. Stermitz F. R., Suess T. R., Fink N. H. and Puzziferri N. (1981) Phytochemical screening of some Rocky Mountain plants. Journal of Natural Products 44, 693± 695. Stoewsand G. S., Anderson J. L., Boyd J. N., Hrazdina G., Babish J. G., Walsh K. M. and Losco P. (1984) Quercetin: a mutagen, not a carcinogen, in Fischer rats. Journal of Toxicology and Environmental Health 14, 105±114. Suzuki S., Takada T., Sugawara Y., Muto T. and Kominami R. (1991) Quercetin induces recombinational mutations in cultured cells as detected by DNA ®ngerprinting. Japanese Journal of Cancer Research 82, 1061± 1064. Watson W. A. F. (1982) The mutagenic activity of quercetin and kaempferol in Drosophila melanogaster. Mutation Research 103, 145±147. Weinstein A. (1936) The theory of multiple-strand crossing over. Genetics 21, 155±199. van der Meer I. M., Stam M. E., van Tunen A. J., Mol J. N. and Stuiteje A. R. (1992) Antisense inhibition of ¯avonoid biosynthesis in petunia anthers results in male sterility. Plant Cell 4, 253±262. Voskresenskii O. N. and Bobyrev V. N. (1978) Eect of ascorbic acid and rutin on the development of experimental peroxide atherosclerosis. Farmakol Toksikol 42, 378±382. Zeiger E. (1993) Mutagenicity of chemicals added to foods. Mutation Research 290, 53±61.
Unaltered Meiotic Chromosome Segregation in Drosophila melanogaster Raised on a 5% Quercetin Diet 1
D. D. SCHRAMM*{1, H. E. COLLINS*2, R. S. HAWLEY2 and J. B. GERMAN1
Department of Food Science and Technology and 2Department of Genetics, Section of Molecular and Cellular Biology, University of California at Davis, Davis, CA 95616, USA (Accepted 1 November 1997)
AbstractÐFlavonoid plant pigments are an integral part of the human diet. Although potentially negative mitotic eects of ¯avonoids have been observed in model organisms, investigation into meiotic eects of ¯avonoids has been neglected. As ¯avonoids aect cell signalling and DNA replication, and because the ¯avonoid content of the human food supply is being increased, determining the eects of ¯avonoids on meiotic ®delity is important. Here, the eect of the human food supply's most prevalent ¯avonoid, quercetin, on the level of meiotic recombination and the amount of X and 4th chromosome non-disjunction in Drosophila melanogaster females was determined. This model organism was chosen since Drosophila melanogaster and Homo sapiens share a remarkable number of commonalties in the meiotic processes of oogenesis and because genetic techniques allow a detailed analysis of meiotic processes in Drosophila. No signi®cant eect on either non-disjunction levels or the percentage distribution of exchange bivalents was observed. A signi®cant eect was observed on the number of ospring; F1 and F2 generations of ¯ies raised on a quercetin diet produced over 10% more progeny than ¯ies raised on a control diet. In this investigation, high quercetin consumption by Drosophila melanogaster females did not pose a threat to meiotic ®delity. # 1998 Elsevier Science Ltd. All rights reserved Keywords: quercetin; ¯avonoid; reproduction; recombination; Drosophila melanogaster; meiosis.
INTRODUCTION
Flavonoids are secondary metabolites synthesized by plants and consumed by humans in fruits, vegetables, legumes, and plant-derived products such as coee, tea, wine and fermented foods (Middleton and Kandaswami, 1993). Human ¯avonoid consumption has been estimated to be in the range of 1 g/day (Kuhnau, 1976), and is increasing for a variety of reasons. First, crop engineers are pursuing increased plant ¯avonoid content with the intent of improving disease resistance in plants and plant nodulation eciency (Dixon et al., 1996; Henkel, 1995). Secondly, ¯avonoids are being considered as potential food additives since they can reduce food spoilage (Grange and Davey, 1990; Nelson, 1982; Ramanathan and Das, 1992; Zeiger, 1993). Lastly, *These authors and their laboratories contributed equally to this work. {Address for correspondence.
corporate marketing has persuaded people to purchase semi-pure ¯avonoids as supplements, using data demonstrating the ability of ¯avonoids to inhibit experimentally induced pathology in animal models (Cook and Samman, 1996; Odetti et al., 1990; Sakaguchi et al., 1992; Voskresenskii and Bobyrev, 1978). Although ¯avonoids inhibit pathology in a variety of experimental systems, potentially detrimental eects of ¯avonoids have been reported. Flavonoids can be mutagenic in the Ames Salmonella test, Drosophila melanogaster recessive sex-linked lethal test, and DNA ®ngerprinting cell culture tests (Bjeldans and Chong, 1977; Suzuki et al., 1991; Watson, 1982). They can disrupt the double helical con®guration of DNA, alter nucleic acid synthesis, and inhibit enzymes such as DNA and RNA polymerase and reverse transcriptase (RT) (Alvi et al., 1986; Austin et al., 1992; Formica and Regelson, 1995; Ono and Nakane, 1990). Furthermore, ¯avo-
0278-6915/98/$19.00+0.00 # 1998 Elsevier Science Ltd. All rights reserved. Printed in Great Britain PII S0278-6915(98)00013-1
586
D. D. Schramm et al.
noids can alter the synthesis and degradation of cell signalling molecules such as the cyclic nucleotides and the eicosanoids. As molecular mechanisms through which ¯avonoids could aect meiotic processes are known and the ¯avonoid content of the human diet is increasing, determining whether ¯avonoids alter meiosis in a detrimental manner is important. Meiotic non-disjunction in humans is the leading cause of human prenatal death and leads to pathology such as Down's syndrome (Sherman et al., 1994). Investigation of possible causes of meiotic non-disjunction in model organisms with similar meiotic processes is an important ®rst step towards understanding the causes in humans. In this experiment, Drosophila melanogaster females were used as model organisms since many aspects of their meiotic process are remarkably similar to those in humans (Koehler et al., 1996). Quercetin was used as a model ¯avonoid since it is the most prevalent ¯avonoid in the human diet and because several mechanisms through which quercetin aects biological processes are known. Females heterozygous for a variety of X chromosome markers were raised on a 5% quercetin diet and then crossed individually to appropriate tester males. The progeny of this cross were analysed to determine the fate of the maternal chromosomes in meiosis. This experimental design allowed identi®cation of eects of quercetin on the amount of progeny produced, the level of meiotic recombination, and the amount of X and 4th chromosome non-disjunction.
MATERIALS AND METHODS
Drosophila melanogaster stocks The X chromosome referred to as MF2 consists of the markers y sc cv v f car, as described previously (Koehler et al., 1996). All chromosome markers are fully recessive and, when homozygous, are scorable by phenotype. Wild-type stock, Oregon RM, was obtained from the Bloomington stock center (Bowling Green, OH, USA). MF2 was kept in stock as MF2/Y C(1)DX/Y, where C(1)DX is an attached chromosome (two X chromosomes physically fused together). The Y chromosome carried a dominant marker Bar of Stone (Bs) to allow identi®cation of XXY females. This experimental design ensured that only primary non-disjunctional events were included in the study (Koehler et al., 1996). Females heterozygous for the MF2 X chromosome and a wild-type X chromosome were crossed to tester males bearing an attached-XY compound chromosome. This attached-XY chromosome was marked with the dominant eye shaped mutation Bar (B) so it could be identi®ed in the presence of another X chromosome.
Food preparation Control food contained the following standard ingredients: 9 g agar (Moorhead and Co., Van Nuys, CA, USA), 40 g nutritional yeast (E.T. Horn, La Mirada, CA, USA), 90 g corn meal, 100 g glucose (Westco, Rancho Cordova, CA, USA), 1 litre water, 3 ml propionic acid (Fisher Scienti®c, Santa Clara, CA, USA), and 2 g n-butyl-hydroxybenzoate (Sigma, St Louis, MO, USA). Ingredients were mixed, boiled, and then cooled, poured into bottles, and allowed to harden. 5 mg yeast was added to the top of each bottle, 2 mg to each vial. Quercetin diets contained control food with 5% quercetin by weight. Quercetin was purchased from Sigma and used without further puri®cation. Quercetin was mixed thoroughly into the food immediately before pouring. The amount of food in control and experimental bottles was equal; food was aliquotted such that all parental ¯ies and progeny larva had access to excess food (i.e. 40 parental ¯ies/bottle; 50 ml food/bottle). The female progeny collected from these bottles were transferred to vials containing 8 ml of control food prepared exactly as for bottles. Experimental design Experimental design is diagrammed in Fig. 1. Virgin wild-type females were crossed to MF2 males and placed in control or 5% quercetin bottles on Day 0. On Day 7, the ¯ies were discarded. The progeny resulting from this mating were collected daily from Day 10 until Day 18. F1 progeny were counted and virgins heterozygous for the maternal chromosome markers were collected. These females, which developed on either control or quercetin diets, were placed individually in vials containing control food and mated to attached-XY tester males for 7 days. Progeny from this mating were counted 15 days after the parental ¯ies were ®rst placed into vials, and male ¯ies were scored for every MF2 marker. As recombination could occur in the mother, males could inherit all MF2 markers, none, or a recombinational product. Male ¯ies expressed all markers inherited from the maternal chromosome since the males contained only a maternal X chromosome. The protocol described allowed determination of the level of recombination in each maternal oocyte. Scoring and statistical analysis Chromosome markers were scored based on their phenotypes, as described previously (Lindsley and Zimm, 1992). Bottles and vials were scored individually, and males were scored separately from females. Unscorable ¯ies (due to broken wings, etc.) were discarded and not included in total counts. Tetrad analysis was calculated as previously described (Weinstein, 1936). Statistical analysis was conducted using Microsoft Excel's (Microsoft Corp., Atlanta, GA, USA) Single Factor Anova,
Meiotic segregation and quercetin
587
Fig. 1. Diagrammatic representation of experimental crosses.
testing the hypothesis that means from samples were equal. In addition, chi-square analysis was performed to determine if the expected (control) and experimental (quercetin) population distributions diered. RESULTS
Eects of quercetin on levels of non-disjunction and mitotic mutation Data on non-disjunction levels in ¯ies raised on control and 5% quercetin diets are presented in Table 1. ANOVA and chi-square analysis indicated no signi®cant dierences between control and quercetin groups. The percentage of non-disjunctional progeny in control ¯ies was 0.08%; the percentage in the progeny of ¯ies raised on the quercetin diet was 0.14%. Eects of quercetin on the percentage distribution of bivalents among the F2 generation The percentage of bivalents with zero (E0), one (E1), two (E2) and three (E3) crossovers is presented in Table 2. Oocytes from female ¯ies raised on a control diet had a tetrad distribution as previously reported (Koehler et al., 1996). Oocytes from females ¯ies raised on a 5% quercetin diet had a similar percentage of bivalents with zero, one or two crossovers. The percentage of E3 bivalents, however, was 0.9% in oocytes from ¯ies raised on the quercetin diet, compared with 0.0% in oocytes
from control ¯ies. This is noteworthy because the normal amount of E3 bivalents found in tetrad analysis of control ¯ies is virtually zero (Merriam and Frost, 1964). ANOVA and chi-square analysis indicated no signi®cant dierences between control and quercetin groups. Eects of quercetin on the number of ospring: MF2 males X wild-type females The eects of dietary quercetin on the number of ospring from Drosophila melanogaster are reported in Table 3. Parent (F0 generation) ¯ies placed on a 5% quercetin diet produced signi®cantly more ospring (F1 generation) than ¯ies placed on the control diet. In addition, female F1 ¯ies removed from bottles with quercetin food, placed on control food, and crossed to attached-XY males raised on control food produced signi®cantly more progeny (F2 generation) than the same cross with ¯ies from control bottles. Quercetin consumption increased male and female progeny equally. In addition, a noticeable but non-signi®cant delay in oocyte development occurred in progeny of ¯ies raised on 5% quercetin diets. Previously, quercetin was observed to induce delays in the development of oocytes from clam and star®sh (Eckberg, 1983). Table 2. Eect of quercetin on the % distribution of bivalents among the F2 generation Exchange ranka E0 E1 E2 E3
Table 1. Eect of quercetin on the amount of non-disjunctional progeny in the F2 generation
Control 5% Quercetin
Total ¯ies
Nondisjunctional progeny
Nondisjunction (%)
5102 5645
4 12
0.08 0.14
Chi-square analysis indicated no signi®cant dierences between control and quercetin groups (P = 0.068).
a
Flies raised on control diet 5.3% 72.4 22.2 0.0
Flies raised on the 5% quercetin diet 8.7%b 68c 22.4d 0.9e
Exchange rank indicates the fraction of chromosomes recovered in each case derived from bivalents with zero (Eo), one (E1), two (E2) or three (E3) exchanges. Tetrad analysis for regular progeny was calculated according to Weinstein (1936). Chi-square analysis indicated no signi®cant dierences between control and quercetin values. bP = 0.348. cP = 0.504. d P = 0.923. eP = 0.534.
588
D. D. Schramm et al. Table 3. Eect of quercetin on the number of Drosophila melanogaster ospring Number of ¯ies/container$
F1 Generation F2 Generation
Containers*
Control
5% Quercetin
% Control
P
5 243
10202 73 8.88 2 6.1
11292 59 10.472 6.3
110.7 117.9
0.03 0.05
*F1 generation ¯ies were raised in bottles and F2 generation in vials. $F1 numbers are the total number of ¯ies in each bottle over eight scoring days and F2 numbers are the number of male ¯ies per scoring day. Statistical analysis was conducted using ANOVA with signi®cance assigned at P < 0.05.
DISCUSSION
In this experiment, dietary quercetin did not signi®cantly alter the rate of meiotic non-disjunction in Drosophila melanogaster females. The percentage of non-disjunction in both control ¯ies and ¯ies raised on a quercetin diet was very similar to the frequency of 0.05% previously reported for similarly marked control ¯ies (Merriam and Frost, 1964). Quercetin's lack of eect on meiotic non-disjunction was unexpected since quercetin is known to inhibit the activity of kinase, polymerase and topoisomerase, disrupt the double helical con®guration of DNA, and alter the synthesis and degradation of cyclic nucleotides involved in the meiotic process (Havsteen, 1983; Middleton and Kandaswami, 1993). In addition to not eecting the amount of nondisjunction, the quercetin diet did not aect tetrad distribution. The trend towards an increase in ¯ies with an unusually large number of exchanges (eE3), from 0.0% in ¯ies on control diets to 0.9% in ¯ies on 5% quercetin diets, suggests the possibility of an eect on meiotic interference. Current theory proposes that the meiotic machinery required for exchange is limited (Loidl, 1994). Thus, if quercetin had increased the amount of meiotic machinery, increased recombination would have been expected; no increased recombination level was observed. In contrast, if interference were reduced, ¯ies with a large number of exchanges could occur with increased frequency without a signi®cant eect on the distribution of exchange. Flavonoids alter a variety of biochemical processes that can impact reproduction in plants and animals. In plants, ¯avonoids are used as sex hormones and can be essential for male gametophyte development (Birch et al., 1953; Burbulis et al., 1996; van der Meer et al., 1992). In insects, ¯avonoids can act as anti- or profeedants, and inhibit or stimulate growth (Hughes, 1988; McFarlane and Distler, 1982; Stermitz, 1981). Here, dietary quercetin increased the number of ospring produced by Drosophila melanogaster. Further investigation should determine whether this increase in ospring is due to increased fertility or increased survival rates. Although the number of ospring from Drosophila melanogaster was increased in this investigation, Stoewsand et al. (1984) noted no increased
progeny from rats raised on quercetin. In addition, oestrogenic ¯avonoids may reduce fertility in vertebrate herbivores (Hughes, 1988). In mammals, therefore, reproduction may not be stimulated by a high quercetin diet. Literature relating the eects of ¯avonoids on mitotic processes in vitro and in vivo is abundant. In contrast, investigation into meiotic eects of ¯avonoids has been neglected. Data from this investigation show only non-signi®cant eects of a 5% quercetin diet on the levels of meiotic recombination and the amount of X and 4th chromosome non-disjunction in ¯ies. This is important, since the meiotic processes of oogenesis in Drosophila melanogaster and Homo sapiens share a remarkable number of commonalties and because non-disjunction is the cause of several genetic diseases in humans including Down's syndrome (Koehler et al., 1996). In addition, although humans currently consume a diet that is less than 1% ¯avonoid by weight, human ¯avonoid consumption is increasing due to crop engineering, the addition of ¯avonoids to food as additives, and through the consumption of puri®ed compounds purchased from health food stores. If the eects of quercetin represent eects of dietary ¯avonoids as a whole and the human meiotic process is aected by quercetin as was that of Drosophila melanogaster, current or increased ¯avonoid intake by humans will not pose a risk to meiotic ®delity. AcknowledgementsÐThis investigation was supported in part by USDA/BARD grant IS2260-93RC. We thank Cora Morgan for editing, Je Sekelsky for technical advice, and Phyllis Wescott for food preparation. REFERENCES
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