In vivo exposure to plant flavonols

Mutation Research, 124 (1983) 255-270 Elsevier

255

MTR 00818

In vivo exposure to plant flavonols Influence on frequencies of micronuclei in mouse erythrocytes and sister-chromatid exchange in rabbit lymphocytes J a m e s T. M a c G r e g o r , C a r o l M. W e h r , G a r y D. M a n n e r s , L e o n a r d J u r d , J a s o n L. M i n k l e r i a n d A n t h o n y V. C a r r a n o U.S. Department of Agriculture, Western Regional Research Center, Berkeley, CA 94710 and I Biomedical Sciences Division, Lawrence Lioermore National Laboratory, Livermore, CA 94550 (U.S.A.) (Received 24 January 1983) (Revision received 16 May 1983) (Accepted 23 June 1983)

Summary No consistent increases in the micronucleus frequency were observed in bone marrow or peripheral blood erythrocytes from mice treated with quercetin, rhamnetin, neohesperidin dihydrochalcone, or hesperetin dihydrochalcone under various exposure and sampling conditions. Over the dose range of 100-1000 mg/kg, quercetin failed to increase significantly erythrocyte rnicronucleus frequencies either (1) in bone marrow of male mice at 6 h after the second of 2 i.p. or oral doses given 24 h apart, or at 48, 96 or 192 h after a single i.p. or oral dose, or (2) in peripheral blood of male or female mice sampled for 7 consecutive days following a single i.p. dose. Feeding 5% or 10% quercetin for 8 days also failed to increase the micronucleus frequency in bone marrow erythrocytes of female or male mice. Hesperetin dihydrochalcone and neohesperidin dihydrochalcone, at p.o. doses of 100-1000 mg/kg, did not increase the micronucleus frequency in bone marrow erythrocytes 6 h after the second of 2 doses 24 h apart, nor did rhamnetin at 48 or 96 h after a single i.p. dose of 1000 mg/kg. Galangin, in contrast, did significantly increase the micronucleus frequency in bone marrow and blood erythrocytes under certain conditions, but the largest increases were only between 2 and 3 times control values and these were observed at highly toxic doses. Rabbits given up to 250 mg/kg quercetin i.p. showed no treatment-related increase in the sister-chromatid-exchange frequency in peripheral blood lymphocytes sampled at 1 and 7 days after treatment. These results fail to confirm published data which report a markedly increased frequency of micronuclei in bone marrow erythrocytes from quercetin-treated mice, show no quercetin-related alterations in the sister-chromatid-exchange frequency in 0165-1218/83/$03.00 © 1983 Elsevier Science Publishers B.V.

256 rabbit lymphocytes, and indicate that clastogenesis in bone marrow erythoblasts due to oral or i.p. administration of the flavonols studied is at most very weak.

The concern over possible genetic effects of dietary flavonoids raised by the first reports of the mutagenicity of quercetin and kaempferol in Salmonella typhimurium (Bjeldanes and Chang, 1977; Sugimura et al., 1977; Hardigree and Epler, 1978) has been increased by subsequent reports of genetic or transforming activity of these and other flavonoids in various types of cells in vitro (Brown and Dietrich, 1979; Brown, 1980; MacGregor and Jurd, 1978; Nagao et al., 1981; Yoshida et al., 1980; Maruta et al., 1979; Umezawa et al., 1977; Amacher et al., 1979; Meltz and MacGregor, 1981; Carver et al., 1983) and in Drosophila (Watson, 1982). Although the major concern is that these agents may pose a hazard to man, the available data on their effects in mammals in vivo is scanty and conflicting. Several dietary carcinogenicity studies have been carried out with quercetin, kaempferol and rutin (quercetin-3rutinoside). These studies are predominately negative (Saito et al., 1980; Hirono et al., 1981; Morino et al., 1982; Ambrose et al., 1952), but Pamukcu et al. (1980) have reported that a dietary level of 0.1% quercetin caused a high frequency of ileal tumors in rats. To our knowledge, the only report of significant genetic damage in a mammalian tissue in vivo is the recent claim of Sahu et al. (1981) that i.p. treatment of mice with quercetin, kaempferol and neohesperidin dihydrochalcone resulted in high frequencies of micronuclei in bone marrow erythrocytes in mice. These results, if confirmed, would provide strong evidence that systemically administered flavonoids were capable of causing either chromosome breakage or anaphase chromosome lag in vivo, and would lend support to the lone positive carcinogenicity report. Since we had previously tested quercetin without observing significantly increased micronucleus frequencies in bone marrow erythrocytes (MacGregor, 1979), we have extended our observations to include a variety of exposure conditions to several flavonoids in order to examine more thoroughly their ability to induce micronuclei. We also include data on the sister-chromatid-exchange (SCE) frequency in the lymphocytes of rabbits exposed to quercetin.

Materials and methods

Flavonoids Quercetin (3,3',4',5,7-pentahydroxyflavone). Concentrated HC1 (150 ml) was added to a suspension of rutin (430 g) in 300 ml methanol and the mixture was refluxed for 2 h. After 30 min, yellow-orange crystals separated from the hot solution. Water (200 ml) was added and the resulting mixture was cooled. Yelloworange crystals were collected, well drained, washed with water and methanol, and air-dried (238 g). The dried solid was boiled with acetone (2 1) and filtered. The filtrate was concentrated (800 ml), ethanol (1 1) was added, and the solution was reconcentrated (800 ml) and cooled to yield quercetin (113 g) as yellow-orange crystals (mp > 300°C, dec.). The ethanol filtrate was further concentrated with an

257

equal volume of water and cooled to produce additional quercetin (186 g). Quercetin (50 g) was recrystallized from tetrahydrofuran/water to yield dark yellow crystals (39 g). HPLC (C18 reverse phase; methanol:water, 55:45) gave a single homogeneous peak which indicated a purity > 95%. In some experiments (Tables 1 and 3) commercial quercetin (Lots 96c and 89c, Sigma Chemical Co., St. Louis, MO) was used. HPLC revealed minor (-4.6%) impurities. Galangin (3,5,7-trihydroxyflavone). ~o-Methoxyphloracetophenone, sodium benzoate and benzoic anhydride were reacted according to the procedure of Kalff and Robinson (1925) to yield 5,7-dihydroxy-3-methoxyflavone, mp 176°C. Acetic anhydride (100 ml) and sodium acetate (20 g) were added to the 5,7-dihydroxy-3methoxyflavone (24 g). The mixture was brought to a boil and heated an additional 30 min on a steam bath. Water (200 ml) and pyridine (0.2 ml) were added and the solution was cooled. The resultant solid was collected and recrystallized from acetone/methanol (2 × ) to yield 5,7-diacetoxy-3-methoxyflavone as slightly brown needles (20 g), mp 172°C. This product was dissolved in acetic anhydride (7 ml). Hydriodic acid (10 ml) was carefully added dropwise and the mixture was refluxed for 5 h. The cooled mixture was poured into water (200 ml), sodium bisulfite (1 g) was added and a cream-colored solid collected. The solid was redissolved in acetone, treated with charcoal, filtered through a celite pad and concentrated with aqueous methanol to yield galangin (3,5,7-trihydroxyflavone) as cream-colored needles (1.3 g), mp 212°C. Found: C, 66.6; H, 3.73. C15Ha005 requires C 66.7; H, 3.73. The synthetic galangin was - 99% pure as determined by an examination of the NMR spectrum. Rhamnetin (7-methoxy-3,3',4,5-tetrahydroxyflavone). Quercetin (5 g), sodium acetate (5 g), and acetic anhydride (20 ml) were boiled for 1 min and poured into water (100 ml). The resulting solid was collected and recrystallized from acetone/methanol to yield quercetin pentaacetate (7.2 g) as colorless needles, mp 199-200°C. Quercetin pentaacetate (20 g), potassium carbonate (50 g), methyl iodide (50 ml) and acetone (300 ml) were refluxed for 72 h. The mixture was filtered, concentrated to 70 ml and methanol (100 ml) was added. Upon cooling, a solid (15 g) was collected. The solid was a mixture of mono- and dimethylated quercetin tetra- and triacetates. The mixed methylated quercetin acetates (15 g) were suspended in ethyl alcohol (200 ml) and concentrated hydrochloric acid (15 ml) and refluxed (15 h). Water (100 ml) was added and the solution was stirred overnight. The solid was collected, washed with water, heated with saturated aqueous sodium borate (300 ml) to boiling and filtered (hot). The filtrate was cooled and acidified, and the resulting solid (5.8 g) was collected. Recrystallization of the solid from tetrahydrofuran yielded rhamnetin (5.0 g) as slightly yellow needles, mp > 300°C, dec. Found: C, 60.6; H, 3.81. C16H120 7 requires: C, 60.8, H, 3.83. The synthetic sample was determined to be of > 95% purity by analytical HPLC (C~8 reverse phase; methanol : water, 55 : 45). Neohesperidin dihydrochalcone (NDHC) was synthesized by Nutrilite Products, Buena Park, CA. The NMR spectrum was consistent with a purity of - 99%. The

258 material employed was the same as that which we supplied to Sahu et al. (1981). Hesperetin dihydrochalcone ( H D H C ) was synthesized from hesperidin by Dr. Robert Horowitz of the USDA Fruit and Vegetable Laboratory, Pasadena, CA, and was subsequently recrystallized from ethanol/water. The purity of H D H C was indicated to be at least 95%. M i c r o n u c l e u s tests in mice

Male or female Swiss-Webster mice with group mean weights at time of initial dosing of 16-32 g (approximately 6 weeks of age) were obtained from Simonsen Laboratories, Gilroy, CA. Animals were age-matched within each experiment. Oral dosing of neohesperidin dihydrochalcone, quercetin and hesperetin dihydrochalcone was by gavage in 2% acacia (gum arabic) in water at 10 m l / k g body weight. Quercetin was also administered by gavage in dimethyl sulfoxide (DMSO) at 5 ml/kg, and by dietary administration at a level of 5% or 10% in a commercial rodent diet (Purina Rodent Chow, Ralston Purina Co., St. Louis, MO). Intraperitoneal (i.p.) administrations of quercetin, galangin, and rhamnetin were in DMSO at 5 m l / k g body weight. In one series of experiments, 375 m g / k g Aroclor 1254 (Monsanto Co., St. Louis, MO) was administered, i.p., to male mice in a volume of 3.75 m l / k g corn oil 4 days prior to treatment with galangin. This series of Aroclor-treated animals was included in the galangin assays because markedly increased in vitro mutagenicity in the Ames Salmonella assay is observed when $9 from mouse or rat liver induced with Aroclor 1254 is employed (unpublished observations). Control animals received either DMSO or acacia by the same route, and in the same solvent volume, as test animals. Triethylenemelamine (TEM, lot 2075-J0710, Lederle Laboratories, Pearl River, NY) and nitrogen mustard (NM, mechlorethamine HCI, lot 42C-2940, Sigma Chemical Co., St. Louis, MO) were given i.p. as positive controls in a volume of saline corresponding to that employed for the test compounds in each experiment. Because we have observed that material injected i.p. may occasionally be delivered to the lumen of the cecum or intestine rather than to the intraperitoneal space, note was made of any animals without residual unabsorbed flavonoid in the peritoneal cavity at the time of sacrifice. Data were analyzed both with and without these animals to be certain dosing artifacts did not influence the results. Three different dose/sampling protocols were employed: (1) Bone marrow was sampled 6 h after the second of 2 doses given 24 h apart as described by Schmid (1976). This is the protocol which was employed by Sahu et al. (1981) in their report of micronucleus induction by quercetin and neohesperidin dihydrochalcone. It is now believed that the second dose is too close to the sampling time to permit any influence on the erythrocyte micronucleus frequency (Cole et al., 1981; Jenssen and Ramel, 1978). Bone marrow from both femurs was suspended in fetal bovine serum and smears were made according to Schmid (1976). Slides were air-dried, fixed in absolute methanol, and stained with filtered Wright-Giemsa stain as described elsewhere (Schlegel and MacGregor, 1982). The incidence of micronuclei in polychromatic and normochromatic erythrocytes, and the ratio of polychromatic to normochromatic erythrocytes, were scored at 1000 x under oil immersion by an observer who was unaware of the identity of the randomized and coded

259 slides. The polychromatic/normochromatic erythrocyte ratio was based on a minimum of 500 erythrocytes. (2) Bone marrow was sampled at various times following a single dose of test agent. Sample preparation and scoring were as described above. Positive control animals were sacrificed at 48 h after treatment, but were dosed at varying times to correspond with actual sampling of animals treated with test compounds. (3) Micronucleus frequencies in peripheral blood erythrocytes were monitored following a single dose of test agent. This protocol is based on recent evidence that micronucleated erythrocytes entering peripheral blood have a lifespan similar to that of normal erythrocytes (Schlegel and MacGregor, 1982) and that the frequencies of micronucleated polychromatic erythrocytes in peripheral blood following single doses of clastogens approximate those observed in bone marrow, but occur about 24 h later (MacGregor et al., 1980). This protocol allows individual animals to be sampled on a daily basis, assuring that the peak incidence in each individual animal is observed. Peripheral blood was obtained by pricking the ventral tail vessels with a 25 G needle, then transferring the blood with a 10-#1 capillary tube to a slide containing 3-5 #1 of fetal bovine serum. The resulting blood smears were treated, stained and scored in the same manner as the bone marrow smears. In all cases, group values are micronucleated cells per total cells scored. The percentage of polychromatic erythrocytes was determined by the number of polychromatic cells in the fields containing the first 500 normochromatic erythrocytes.

Sister chromatid exchange in rabbit lymphocytes New Zealand White male rabbits, approximately 4 kg body weight, were injected i.p. with DMSO (2.5 ml) or quercetin at 250 mg in 0.63 ml of DMSO, 500 mg in 1.25 ml DMSO or 1000 mg in 2.5 ml of DMSO. This concentration of quercetin (400 m g / m l ) was near the limit of solubility. Approximately 2 ml of blood were withdrawn by nicking the marginal ear vein of each animal prior to, 1 day after, and 7 days after each injection. Blood was cultured according to previously described methods (Stetka et al., 1978). Briefly, whole blood cultures were established in minimal essential medium with 10 #M bromodeoxyuridine and phytohemagglutinin. Approximately 44 h after the cultures were initiated, colcemid (0.1 # g / m l final concentration) was added for 4 h and the cells were harvested for slide preparation. The metaphase chromosomes were stained to differentiate the sister chromatids according to the procedure of Perry and Wolff (1974) as modified by Minkler et al. (1978). A minimum of 20 cells were scored per animal at each sampling time. Statistical analyses Micronucleus frequencies in polychromatic erythrocytes of treated groups were compared with concurrent control values using both the negative binomial comparison described by Mackey and MacGregor (1979) and the binomial comparison described by Kastenbaum and Bowman (1970). Micronucleus frequencies in normochromatic erythrocytes were compared with concurrent control group values by the binomial comparison of Kastenbaum and Bowman (1970). Values which exceed the critical value for significance at the 5% or 1% levels are

26O TABLE 1 INCIDENCE OF MICRONUCLEATED ERYTHROCYTES 1N BONE MARROW OF MALE MICE EXPOSED TO PLANT FLAVONOLS ~ Dose (mg/kg)

N

Micronucleated PCE/1000 PCE

Micronucleated NCE/1000 NCE

PCE (%)

Quercetin, i.p. 1000 DMSO NM, 5

12 11 11

1.2 1.6 25.0 tt'**

(12078) (11096) (8755)

0.2 0.5 0.8

(20432) (14068) (17105)

41 48 44.

Galangin, i.p. 1000 300 DMSO NM, 5

8 8 8 7

1.4 1.2 1.2 53.0 tt'**

(4147) (4111) (4076) (3585)

1.7 1.6 0.9 1.9

(4210) (4321) (4214) (3768)

27 23* 31 27

13

Aroclor + galangin, i.p. 1000 9 300 8 100 8 Aroclor/DMSO 8 NM, 5 7

1.3 1.0 1.7 2.2 53.0 tt'**

(4667) (4155) (4153) (4088) (3585)

1.0 1.4 3.6* 1.2 1.9

(4897) (4235) (4461) (4144) (3768)

17' 14'* 16'* 31 27

42 22 11

Pooled controls from above i.p. experiments 19 1.5 (15172) DMSO 18 33.0 tt'** (12340) NM, 5

0.6 1.0

(18 282) (20873)

40 36

Quercetin, p.o. 1000 500 247 100 2% acacia TEM, 0.25

(6039) (6142) (6069) (6162) (12296) (11740)

0.5 0.7 0.9 0.0 0.7 1.6

(3687) (2795) (6361) (2382) (7628) (7044)

62 69 49 72 62 62

Neohesperidin dihydrochalcone, p.o. 5000 6 1.3 1000 6 0.5 500 6 3.1N'* 500 6 1.4 200 6 0.3 2% acacia 12 1.1 TEM, 0.25 12 16.0 tt'**

(6152) (6056) (6136) ¢ (6 399) (6126) (12413) (11736)

2.5 0.4 1.5" 0.3 0.0 0.6 1.3

(4363) (2829) (4694) e (3 435) (2211) (7135) (6 923)

59 68 57 65 73 64 62

Hesperetin dihydrochalcone, p.o. 1000 6 1.0 300 6 1.0 100 6 1.8 2% acacia 6 0.8 TEM, 0.25 6 24.0 tt'**

(6228) (6102) (6109) (6095) (6133)

1.0 0.3 0.3 0.3 1.6

(3 829) (3430) (3201) (3142) (3060)

62 64 66 66 67

Pooled controls from above p.o. experiments 2% acacia 24 0.9 (24571) TEM, 0.25 24 20.0 tt'** (23985)

0.7 1.5

(13268) (13627)

65 64

6 6 6 6 12 12

1.3 1.3 0.7 0.8 1.0 18.0 it'**

Mortality b (%)

a Swiss-Webster mice (20-32 g) were given 2 doses 30 and 6 h prior to sacrifice. b Mortality is zero unless otherwise noted. c Only 1 animal of the group was affected; group value is not significant if this animal (14 micronucleated PCE/1000 PCE, 3 micronucleated NCE/245 NCE) is not included.

261 TABLE 1 (continued) Statistical comparisons were made as described in Materials and methods. Numbers of cells uPon which each micronucleus frequency is based are given in parentheses. Significance levels of groups differing from the concurrent control are as follows: negative binomial: t denotes P < 0.05, tt denotes P < 0.01, N indicates no decision possible at a = 0.05, fl = 0.1; binomial comparison: * denotes P < 0.05, ** denotes P < 0.01; Kruskal-Wallis analysis of variance, where comparison among all dosage groups and concurrent control was significant at P < 0.05, individual dose groups subsequently found to differ from their concurrent control are indicated * where P < 0.05 and *~; where P < 0.01.

i n d i c a t e d for e a c h test in the tables a n d figures. T h e n e g a t i v e b i n o m i a l test w a s set u p to d e t e r m i n e w h e t h e r o r n o t a 3-fold or g r e a t e r i n c r e a s e o v e r the s p o n t a n e o u s v a l u e in all c o m p a r a b l e c o n t r o l g r o u p s was o b s e r v e d at a t y p e 2 ( f l ) e r r o r o f 0.10 ( M a c k e y a n d M a c G r e g o r , 1979). T h e K r u l k a l - W a l l i s 1 - w a y a n a l y s i s o f v a r i a n c e b y r a n k s (Siegel, 1956) w a s u s e d to test for d i f f e r e n c e s in the p e r c e n t a g e o f p o l y c h r o m a t i c e r y t h r o c y t e s a m o n g g r o u p s . W h e n the a n a l y s i s o f v a r i a n c e for the c o n c u r r e n t n e g a t i v e c o n t r o l a n d all d o s a g e g r o u p s of a single test a g e n t was s i g n i f i c a n t at P < 0.05, s u b s e q u e n t p a i r w i s e c o m p a r i s o n s w e r e m a d e to d e t e r m i n e w h i c h i n d i v i d u a l d o s a g e g r o u p s d i f f e r e d f r o m the concurrent control group. S i s t e r - c h r o m a t i d - e x c h a n g e f r e q u e n c i e s in t r e a t e d a n i m a l s w e r e c o m p a r e d w i t h D M S O c o n t r o l v a l u e s u s i n g S t u d e n t ' s t test.

. o ~ , ~ , , . ° ~ °" U~,,,~o. "-'°" HO

0 QUERCETIN

Me O ~ / ~ y ~ O ~

~

HO

0 H~"~e',- 0 H

HO

0

OH

0 RHAMNETIN

GALANGIN

,,_G,o~O" l . ~ -°M" "OH HO

HO

O HESPERETIN DIHYDROCHALCONE

Fig. 1. Structures of compounds investigated.

0

NEOHESPERIDIN DIHYDROCHALCONE

263 at 4 days after a single i.p. dose of I g / k g quercetin when the statistical analysis was made by multiple single-group comparisons with the concurrent control group, but this result was not reproduced in a repeat experiment. Galangin, in contrast, significantly increased the frequency of micronuclei at both 2 and 4 days after i.p. treatment. Similar elevations in the micronucleus frequency occurred in mice given Aroclor 1254 4 days prior to receiving galangin, but the increase was statistically significant only at the 2-day sampling period, due to the reduced sample size at day 4 which resulted from the higher mortality and polychromatic cell suppression in the Aroclor-treated mice. Rhamnetin, given i.p. at 1000 m g / k g , did not significantly alter the micronucleus frequency. Quercetin and rhamnetin both caused a slight reduction in the polychromatic cell frequency at 48 h after a dose of 1000 m g / k g i.p. as well as a low incidence of mortality in the 2-, 4- and 8-day treatment groups.

A 45 L

**~tt

o.o

/1

G.

NITROGEN MUSTARD,2.S mglkg n = 5

r I ~,/ '° 1-

\\

,,ol

\

/

**~tt

5F /

~,,,.,°°°°.,,.... ~. ° ° ,oo,.,,,.N ,,oo.,..... 6

~ DMSO,$mllkg, n=7

**Ott

**r~t

.~[

....

,r-'~,~._~

i~1. . . . .

"[3-.

N

,~,J...-'~,~- - = ~ ~ _ ~

/ o 120

B

I 1

, 2

o** 3

"" . . . . . . 4

~o 5

I 6

l 7

DAYS Fig. 2. Effect of galangin and quercetin on the micronucleus frequency in peripheral blood erythrocytes. (A) Micronucleus frequency in polychromatic cells foUowing intraperitoneal injection of quercetin or galangin. No significant changes were observed in normochromaticcells, u N = 6 on day 2; 5 on day 3; 1 on subsequent days. (B) Percentageof polychromaticcells. Value for each animal is based on a minimum of 500 cells. Error bars are the S.E.M. See Table 1 for key to statistical analyses.

Aroclor + galangin, i.p. 1000 100 33 Aroclor + DMSO, i.p. NM, 5

Galangin, i.p. 1000 333 100 DMSO, i.p. NM, 5 4.6 N,,

1.7 56.0 tt'**

8 22 c

1.4 56.0 tt'**

8 22 c

5

3.0r~, **

1.7

1.6

1.2 0.5 70.0 tt'**

(4113) (10714)

(2582)

(4166) (10714)

(3613)

(4175)

(3125)

(4139) (4154) (6210)

Micronucleated PCE/1000 PCE

7

8

6

Rhamnetin, i.p. 1000

Quercetin, i.p. 1000 1000 333

8 8 14

N

Day 2

Quercetin, p.o. 1000 DMSO, p.o. NM, 5

Dose (mg/kg)

(3722)

(4303)

(3107)

(3534) (3382) (7766)

(2 889)

2.0 (4009) 9.5** (13432)

1.4

0.3 (2947) 9.5** (13432)

0.8

1.2

1.0

1.1 0.9 8.8**

Micronucleated NCE/1000 NCE

45 20 ¢¢

31

59 20 ts

34 ss

45 ~;*

49 ~

49 55 20 ¢~

PCE (%)

17

(%)

Mortality b

INCIDENCE OF MICRONUCLEATED ERYTHROCYTES IN BONE MARROW OF MALE MICE AT VARIOUS TIMES AFTER EXPOSURE TO PLANT FLAVONOLS a

TABLE 2

8

6 8 8

8 4 8 16

Rhamnetin, i.p. 1000

Quercetin, i.p. 1000 1000 333

Galangin, i.p. 1000 333 100 DMSO, i.p.

3.1

(7649)

(1406) (2040) (1030)

(8202) (5143) (5648) (17545)

(3139) (4086) (4067)

(4125)

(4106) (4112)

0.8

2.4 2.8 2.0

0.7 3.4"* 2.4* 0.8

1.2 1.1 1.1

1.0

0.9 2.2

(7218)

(2105) (2132) (1018)

(8460) (5599) (5091) (15087)

(2444) (3555) (3543)

(3972)

(3474) (3217)

Micronucleated NCE/1000 NCE

47

7 *) 29 47

26 *$ 32 39 50

50 51 50

46

54 56

PCE (%)

(%)

6d

78 69 50

50 56 20

25

11

8

7

7

N Mortality b

(4583)

(3634)

1.7

3.0 N

(4586)

5.0 t t

Micronucleated PCE/1000 PCE

Day 8

Swiss-Webster mice (20-32 g) were given a single dose at specified times prior to sacrifice. Mortality is zero unless otherwise noted. Pooled animals from i.p. experiments. 7 out of 7 additional animals receiving Aroclor 1254 but no D M S O survived at least 1 month.

15

6.4 N 5.4 N 1.9 N

3.4 N'** 5.4 N'* 3.4 r~ 2.2

5.4 t'* 2.4 1.5

1.7

3.4 ~ 2.7

Micronueleated P C E / 1 0 0 0 PCE

See Table 1 for key to statistical analyses.

a b ¢ d

Aroclor + DMSO, i.p.

Aroelor + galangin, i.p. 1000 2 100 4 33 2

8 8

Quereetin, p.o. 1000 DMSO, p.o.

N

Day 4

(continued)

Dose ( m g / k g )

TABLE 2

PCE

(4214)

51

46 (3406) 1.5

1.4

41 (5103)

(%)

0.8

Micronucleated NCE/1000 NCE

13

(%)

Mortality b

to

Day 4

6 7

Quercetin 1000, i.p. DMSO, i.p.

0.7 (3052) 0.9 (3538)

1.4 (3585) 1.5 (4115)

Micronucleated P C E / 1 0 0 0 PCE

0.6 (3111) 0.6 (3621)

1.1 (3644) 0.9 (4254)

Micronucleated NCE/1000 NCE

0.6 (3545) 0.8 (3573)

1.7 (4042) 0.7 (4068)

Micronucleated P C E / 1 0 0 0 PCE

3.8 3.8

4.8 6.8 b

PCE (%)

2.0 (3573) 0.6 (3588)

1.0 (4183) 1.7 (4104)

25.0 12.5

12.5 _

(%)

Mortality

Micronucleated NCE/1000 NCE

6 7

7 8

N

1.3 1.4

0.8 2.0 N

(3073) (3597)

(3621) (4090)

Micronucleated P C E / 1 0 0 0 PCE

Day 8

4.4 4.1

3.1 ~; 5.4 b

PCE (%)

1.0 (3133) 2.5 (3666)

1.7 (3583) 1.0 (4174)

Micronucleated NCE/1000 NCE

12.5 12.5

(%)

Mortility

4.3 4.3

7.0 4.3

PCE (%)

25.0 12.5

12.5

(%)

Mortality

See Table 1 for key to statistical analyses.

a Swiss-Webster female mice (16-22 g) were given a single dose; tail blood was sampled on successive days. Initial group size was 8 animals. b 1 animal was excluded from group as an outlier for ratio determinations; values excluded were 18.5% on day 2, 37.4% on day 4.

7 8

Quercetin 1000, p.o. DMSO, p.o.

N

7 7

Quereetin 1000, i.p. DMSO, i.p.

Dose ( m g / k g )

8 8

N

Day 2

Quercetin 1000, p.o. DMSO, p.o.

Dose ( m g / k g )

I N C I D E N C E O F M I C R O N U C L E A T E D E R Y T H R O C Y T E S IN P E R I P H E R A L B L O O D OF F E M A L E MICE U P T O 8 DAYS A F T E R A SINGLE DOSE O F Q U E R C E T I N , O R A L O R i.p. a

TABLE 3

to

267 TABLE 4 INCIDENCE OF MICRONUCLEATED ERYTHROCYTES IN BONE MARROW OF MALE OR FEMALE MICE ON DIETS CONTAINING 5 OR 10% QUERCETIN FOR 8 DAYS a Diet

N

Micronucleated PCE/1000 PCE

Micronucleated NCE/1000 NCE

PCE (%)

10 10 9

1.0 (5136) 1.2 (5197) 1.1 (4610)

0.8 (5136) 1.2 (5166) 0.9 (4627)

62' 69** 55

10 10 10

1.0 (5 094) 1.9 (5 318) 1.0 (5154)

0.8 (5 090) 1.2 (5132) 0.8 (5 206)

60 * 65** 51

Males

Quercetin 10% Quercetin 5% Purina control Females

Quercetin 10% Quercetin 5% Purina control

a Swiss-Webster mice (16-21 g) were fed diets ad lib for 8 days. See Table 1 for key to statistical analyses.

Galangin caused significant mortality at all doses tested and significantly depressed the polychromatic cell frequency at doses of 100 mg/kg or above. The above results with quercetin and galangin are supported by the experiment summarized in Fig. 2, in which the micronucleus frequency in peripheral blood was monitored each day for 7 days after a single i.p. dose of quercetin or galangin. In agreement with the bone marrow data, quercetin failed to produce a significant increase, while galangin induced a significant increase at 48 h, with a similar, but not statistically significant, increase at 72 h. Because a 10-fold increase in the frequency of micronucleated polychromatic erythrocytes in the bone marrow of female, but not male, mice fed 10% quercetin in the diet for 1 week was recently reported *, we have also examined the micronucleus frequencies in bone marrow erythrocytes from both female and male mice fed quercetin in a commercial diet (Purina Rodent Chow) for a period of 8 days at levels of 5% and 10%. No significant increases were observed in either the polychromatic or normochromatic erythrocytes under these conditions (Table 4). Results of the rabbit lymphocyte SCE experiments are given in Table 5. No significant differences due to quercetin were observed. Thus, quercetin failed to induce significant increases in either SCE or micronucleus frequencies under any experimental condition examined. Among the compounds and conditions examined, the increase in micronucleus frequency induced by galangin was the only significant response observed. Our interpretation of this increase is somewhat cautious because the increases observed were relatively small and were only observed at highly toxic doses of galangin. Galangin was much more toxic than the other flavonoids tested, and animals * Verbal report by M. Nagao, Symposium on Mutagens and Carcinogens in Food, Kyoto Satellite Meeting of Third International Conference on Environmental Mutagens, Kyoto, Japan, September 26-27, 19811

268 TABLE 5 THE FREQUENCY OF SCE IN LYMPHOCYTES OF RABBITS INJECTED WITH QUERCETIN Day

DMSO

0a 1 7

5.0±0.6 b 5.9±0.3 8.8±1.2 ¢

Dose (mg) 250

500

1000

5.6±0.4 5.3±0.7 6.0±0.2

5.0±0.2 5.9±0.2 8.2±1.2

4.3±0.1 4.8±1.0 6.1±0.6

a Blood samples were drawn prior to injection. b Mean and standard error of the individual rabbit means. c Significantly increased compared to day 0 control rabbits for that treatment (p < 0.05, Student's t test).

e x h i b i t i n g i n c r e a s e d m i c r o n u c l e u s frequencies were frequently a l r e a d y m o r i b u n d at the time o f sampling. T h e relatively weak response o b t a i n e d with g a l a n g i n m a y be c o m p a r e d with that o b t a i n e d with a typical k n o w n clastogen, n i t r o g e n m u s t a r d , in Fig. 2 a n d T a b l e 2. W h e t h e r this weak effect of g a l a n g i n is due to direct g e n o t o x i c i t y o r is s e c o n d a r y to the m a r k e d toxicity c a n n o t be resolved w i t h o u t a d d i t i o n a l m e c h a n i s t i c experiments.

Acknowledgements A p o r t i o n of this w o r k was p e r f o r m e d u n d e r U S D A c o n t r a c t N o . 5 3 - 9 A H Z - 8 - 1 4 4 3 a n d u n d e r the U.S. D e p a r t m e n t o f E n e r g y C o n t r a c t N o . W-7405-Eng-48. Reference to a c o m p a n y a n d / o r p r o d u c t n a m e d b y the U.S. D e p a r t m e n t of A g r i c u l t u r e or the U.S. D e p a r t m e n t o f E n e r g y is o n l y for p u r p o s e s of i n f o r m a t i o n a n d does n o t i m p l y a p p r o v a l o r r e c o m m e n d a t i o n of the p r o d u c t to the exclusion of others which m a y also be suitable.

Note added in proof T w o relevant p u b l i c a t i o n s have a p p e a r e d since p r e p a r a t i o n of this m a n u s c r i p t . A e s h b a c h e r et al. ( N u t r i t i o n a n d Cancer, 4 (1982) 9 0 - 9 8 ) r e p o r t failure to d e m o n strate increased m i c r o n u c l e u s frequencies in the b o n e m a r r o w of male mice given p.o. doses o f quercetin from 1.0 to 1000 m g / k g , a n d C e a et al. ( M u t a t i o n Res., 119 (1983) 3 3 9 - 3 4 2 ) r e p o r t i n d u c t i o n of m i c r o n u c l e i in m o u s e b o n e m a r r o w e r y t h r o c y t e s after i.p. t r e a t m e n t with 5,3',4'-trihydroxy-3,6,7,8-tetramethoxyflavone.

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