Lactational transfer of the soy isoflavone, genistein, in Sprague–Dawley rats consuming dietary genistein

Reproductive Toxicology 21 (2006) 307–312

Lactational transfer of the soy isoflavone, genistein, in Sprague–Dawley rats consuming dietary genistein Daniel R. Doerge a,∗ , Nathan C. Twaddle a , Mona I. Churchwell a , Retha R. Newbold b , K. Barry Delclos a a

Division of Biochemical Toxicology, National Center for Toxicological Research, U.S. Food and Drug Administration, Jefferson, AR 72079, USA b Developmental Endocrinology and Endocrine Disrupter Section, Laboratory of Molecular Toxicology, National Institute of Environmental Health Sciences, NIH/DHHS, Research Triangle Park, NC 27709, USA Received 8 July 2005; received in revised form 15 August 2005; accepted 13 September 2005 Available online 28 October 2005

Abstract Exposures of Sprague–Dawley rats to the soy isoflavone, genistein, throughout the entire lifespan have produced a number of effects on reproductive tissues, immune function, neuroendocrine function and behavior. Our previous studies investigated pharmacokinetics and disposition of genistein during adult and fetal periods and this study describes the internal exposures of post-natal day 10 (PND10) rat pups due to lactational transfer of genistein. Conjugated and aglycone forms of genistein were measured by using LC/MS/MS in serum (PND10) and milk (PND7) from lactating dams consuming a genistein-fortified soy-free diet, and in serum from their pups at a time when milk was the only food source (PND10). This study shows that limited lactational transfer of genistein to rat pups occurs and that internal exposures to the active aglycone form of genistein are generally lower than those measured previously in the fetal period. These results suggest that developmental effects attributable to genistein exposure in our chronic and multi-generation studies are more likely to result from fetal exposures because of the higher levels of the active estrogenic aglycone form of genistein in utero, although the possibility of neonatal responses cannot be excluded. © 2005 Elsevier Inc. All rights reserved. Keywords: Genistein; Soy; Lactation; Neonatal; Mass spectrometry

1. Introduction Multi-generation reproductive and chronic toxicity studies of genistein have been conducted by the National Toxicology Program (NTP) at this institution, where Sprague–Dawley rats were exposed to genistein during all life stages. In the dose range-finding studies, rats were exposed to dietary doses of genistein up to 1250 ppm from gestational day 7 into adulthood. Effects were observed in both males and females including hyperplasia of mammary glands in both sexes, aberrant or delayed spermatogenesis, histological changes in the vagina and ovary, mineralization of renal tubules in males [1], modulation of natural killer cell activity [2], myelotoxicity [3], neuroendocrine changes associated with behavioral outcomes

∗

Corresponding author. Tel.: +1 870 543 7943; fax: +1 870 543 7720. E-mail address: [email protected] (D.R. Doerge).

0890-6238/$ – see front matter © 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.reprotox.2005.09.007

[4,5], and sexually dimorphic brain development [6]. Genistein exposures through placental transfer, lactational exposure and ingestion of dosed feed could all have contributed to the observed effects. Exposure to genistein aglycone and its Phase II conjugates (glucuronides and sulfate) was previously evaluated in serum and organs of adult rats and in the serum during the post-wean period when rats were ingesting genistein-fortified diets [7]. In adult rats consuming 500 ppm dietary genistein, the high dose utilized in the multi-generation and chronic studies that followed the dose range-finding studies, total circulating genistein reached levels of approximately 6–8 ␮M, with conjugated forms accounting for 95–99% of the total. Tissue/serum partition coefficients ranged from 0.05 to 0.2 for total and from 0.6 to 26 for aglycone genistein, consistent with significant tissue exposures to the active estrogenic form [7]. In a subsequent study, exposure to genistein was also determined during gestation when rat fetuses were exposed via

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placental transfer [8]. Genistein was detected in fetal plasma and brain 2 h after orally dosing pregnant rats. Total genistein levels were lower in fetal plasma than in the plasma of the dams, although the percent present as aglycone was higher (approximately 30%) in the fetus, possibly due to the less efficient glucuronidation capacity of the fetus. This higher aglycone exposure and possible differences in blood–brain barrier resulted in brain levels of aglycone genistein that were approximately five-fold higher than a correspondingly exposed adult. These findings suggested that significant exposures to the active form of genistein could occur during the fetal period. The early post-natal period is also a critical time in the development of the reproductive system of rodents [9,10] and chemical exposure during this period has been widely used to model the effects of diethylstilbestrol and other potential endocrine disruptors in humans [9,11,12], including genistein [13–18]. Because of the sensitivity of this exposure period, it was important to measure directly the levels of genistein transferred to pups via milk under conditions where residual genistein from placental transfer would not be an issue but where the pups were not yet old enough to consume solid food. Limited and conflicting information on the transfer of genistein through lactation is available. While the transfer of genistein to human breast milk has been reported in a woman following consumption of 0.08–0.33 mg/(kg bw) genistein in roasted soy nuts, the level of aglycone genistein was below the detection limit [19,20]. The total genistein present in milk was only 7% of that in plasma. Prior to the start of our study, the data of Fritz et al. [21] were the most relevant to lactational exposure of Sprague–Dawley rats to genistein. While lactating dams fed 250 ppm genistein in AIN-76A diet had serum total genistein levels of 0.42 ␮M, their post-natal day (PND) 7 pups were reported to have serum levels of 0.73 ␮M. Milk from the dams contained 0.14 ␮M of total genistein but the pup stomach contents reportedly contained 4.4 ␮M. Stomach contents and pup serum contained genistein aglycone at percentages greater than 75 and 14%, respectively, and were considerably higher than the aglycone percentages found in the milk (57%) or serum of dams (2%). Thus, it appeared that genistein aglycone was present in rat milk at high concentrations and was efficiently transferred to the serum of pups. Weber et al. [22] also reported high concentrations of total isoflavones in the plasma of infant rats and concluded that there was efficient transfer of genistein from the mother’s plasma to milk. On the other hand, a more recent study by Lewis et al. [23] reported little transfer to the milk of dams administered a single dose of 50 mg genistein/(kg bw) by gavage. Peak concentrations of total genistein in milk (0.63 ␮M) were approximately 9% of those in dam plasma (6.7 ␮M). In our previous study [8], the serum of 1–2-day-old pups of dams fed 500 ppm genistein contained 0.176 ␮M total genistein, of which approximately 53% was the aglycone. However, in these pups, the measured genistein may have reflected residual genistein from placental transfer as well as from suckling. This study was conducted to define the internal exposures of pups to genis-

tein during the early neonatal lifestage in our multi-generation studies. 2. Materials and methods 2.1. Reagents Genistein, with purity greater than 99% was obtained from Toronto Research Chemicals (Ont., Canada); glucuronidase/sulfatase from Helix pomatia containing 105 units/ml glucuronidase activity + 5 × 103 units/ml sulfatase activity and oxytocin were obtained from Sigma Chemical Co. (St. Louis, MO). Deuterated genistein (6,8,3 ,5 -d4, 95%) was purchased from Cambridge Isotope Laboratories (Andover, MA) and characterized previously [7]. All solvents were HPLC grade and Milli-Q water was used throughout.

2.2. Animal handling procedures All procedures involving care and handling of animals were reviewed and approved by the NCTR Institutional Animal Care and Use Committee. Ten plug-positive female CD (Sprague–Dawley) rats were from the NCTR colony. Rats were maintained on a soy-free basal diet consisting of irradiated 5K96 meal (Purina Mills Inc., St. Louis, MO) for 2 weeks prior to mating. This commercially available diet, which is produced in Purina’s test diet facility, is similar to the standard NIH 31 rat feed except that the soymeal and alfalfa components are replaced by casein, soy oil is replaced by corn oil and the vitamin content is adjusted to compensate for irradiation effects. The genistein (0.54 ␮g/g) and daidzein (0.48 ␮g/g) content in this feed was determined using LC–ES/MS/MS analysis after complete hydrolysis of glucoside conjugates. Immediately after delivery, nursing rats were allocated to either the control 5K96 diet or a genistein-fortified diet (500 ppm algycone in 5K96). The concentrations of genistein in diets were verified using LC–UV (260 nm detection; data not shown). Litters were culled to eight pups per litter (equal numbers of males and females). Total feed consumption by the dams was determined for the 10 days exposure period by daily measurement of feeder box weights and was not corrected for possible spillage. Dam body weights and total litter weights were recorded at birth, PND7 and 10. Dams were removed from their litters and administered oxytocin (4 IU/(kg bw) in 1 ml/(kg bw)) by intraperitoneal injection [24]. Approximately 5 min later, milk was collected from all dams on PND7 by applying an eye dropper to the nipple area and aspirating a small volume of milk (ca. 100 ␮l) from each dam that was stored at −80 ◦ C until analyzed. Dams were immediately returned to their litters upon completion. The exposure was concluded on PND10, a time when the pups had not yet begun to consume solid food, with sacrifices conducted in the early morning. Dams and pups were asphyxiated using CO2 and blood was removed by cardiac puncture for preparation of serum that was stored at −80 ◦ C until analyzed. Serum was collected separately for each dam and from individual pups in every litter.

2.3. Serum and milk genistein analysis Rat serum and milk from individual dams and their pups was analyzed for both total and aglycone genistein using an LC/MS/MS method previously validated [25] Briefly, a 10–100 ␮l aliquot of serum was thawed, deuterated genistein internal standard was added, an equal amount of acetonitrile was added to precipitate proteins, the mixture was centrifuged, a 100 ␮l aliquot of the solution was diluted with 0.4 ml citrate buffer (25 mM, pH 5.0) with or without deconjugating enzymes, and a 450 ␮l aliquot was purified using automated solid phase extraction and analyzed by using tandem mass spectrometric detection for isotopedilution quantification of genistein. The milk was similarly analyzed (25 ␮l aliquots) after initial method validation performed using milk obtained from untreated lactating rats (data not shown). The limit of detection (LOD) for genistein from analysis of 100 ␮l of serum was 0.0005 ␮M and in milk was 0.006 ␮M. The precision of measurements was in the range of 4–8% relative standard deviation (COV) and the corresponding accuracies were 88–96% from samples between 0.02 and 10 ␮M [26]. Quality control procedures included concurrent analysis of control rat serum (soy-free diet), genistein-spiked control serum and a

D.R. Doerge et al. / Reproductive Toxicology 21 (2006) 307–312 mixture of labeled and unlabeled standards interspersed throughout each sample set.

2.4. Liquid chromatography and mass spectrometry Liquid chromatography (LC) separations were performed using a 2795 liquid handling system (Waters, Milford, MA). Chromatography was performed using an Ultracarb ODS column (either a 2 mm × 30 mm, 3 ␮m particle size, Phenomenex, Torrance, CA) with isocratic elution (65% of aqueous 0.1% formic acid/35% of acetonitrile). The flow rate was 0.3 ml/min and the entire effluent was introduced into the electrospray (ES) probe of a Quattro Ultima triple quadrupole mass spectrometer (Waters, Manchester, UK) with an ion source temperature of 150 ◦ C. The method monitored MRM transitions for d0- and d4-geinstein (m/z 271 → 215 and 275 → 219, respectively) using a dwell time of 0.3 s, sampling cone-skimmer potentials of 25 V, Ar gas cell pressures of 1.1 × 10−3 mbar, and collision energy of 27 eV.

2.5. Statistics Statistical significance was assessed using either the t-test (two-sided) or linear regression analysis with p < 0.05 as the criterion.

3. Results Exposures to genistein were evaluated in serum from lactating dams on PND10 (Table 1). There was no significant difference in body weights between the genistein-exposed and control rats. Levels observed in rats maintained on the soy-free control diet consistently had serum genistein levels below method detection limits (LOD < 0.0005 ␮M). In rats fed the genisteinfortified diet, serum total genistein was observed in the range of 0.15–2.99 ␮M. The percentage of aglycone genistein in dam serum was in the range of 1.7–2.4% (mean 2.4 ± 0.7, n = 4), with an outlier (#7) at 17% (>3S.D. from mean). Concentrations of aglycone were not significantly correlated with total genistein in dam serum (r2 = 0.22, p = 0.43). This large range in serum concentrations was not reflected by the dietary doses consumed, which were quite consistent (51 ± 1.8 mg/(kg bw day), Table 1). This range presumably reflects heterogeneity in the timing of feed consumption by the rats prior to sacrifice coupled with the

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fast elimination of genistein by rats (4.3 ± 0.29 h in non-lactating female rats [7]). Milk was sampled after administration of oxytocin to stimulate release from the dam. This procedure has been used extensively to collect milk [24] and has been shown to have minimal effects on rat pup growth when administered throughout the nursing period [26]. Also, normal nursing behavior has been reported in dams consuming genistein [27]. Milk was collected 3 days prior to sacrifice in order for the putative steady state concentration to be re-established well before sampling of serum (see below). Milk collected on PND7 from control rats consistently showed genistein levels below detection limit (0.006 ␮M). Milk from rats consuming the genistein-fortified diet contained total genistein in the range of 0.28–0.81 ␮M with associated aglycone levels that were 18–52% of the total (Table 2). No significant relationship was observed between total or aglycone genistein concentrations in dam serum and the respective concentrations in milk (r2 = 0.18, p = 0.48; r2 = 0.09, p = 0.62, respectively). Serum collected on PND10 from control pups consistently had serum genistein levels below method detection limits (0.0005 ␮M; Table 2). Serum from pups consuming milk from genistein-fed dams contained litter mean values for total genistein levels in the range of 0.022–0.053 ␮M with associated aglycone levels that were 1.2–4.6% of the total (Table 2). No significant sex differences were observed so all data shown are combined from male and female pups. The ratio of litter mean pup serum genistein values to dam serum genistein values ranged between 0.01 and 0.28 for total and between 0.01 and 0.15 for aglycone with respective means of 0.12 ± 0.12 and 0.07 ± 0.08. No significant relationship was observed using linear regression analysis between litter mean values for total or aglycone genistein concentrations in pup serum and the respective concentrations in milk (r2 = 0.06, p = 0.90; r2 = 0.28, p = 0.36, respectively) or dam serum (r2 = 0.68, p = 0.09; r2 = 0.04, p = 0.24, respectively). Body weight gain over the PND7–10 period was significantly lower for male and female pups suckling on genistein-fed dams

Table 1 Genistein exposure data and estimates of dosing from lactating Sprague–Dawley dams Litter #

BW (g)

Feed consumption (g/day)

1-Control 2-Control 3-Control 4-Control 9-Control 5-Genistein 6-Genistein 7-Genistein 8-Genistein 10-Genistein

276.9 331.8 278.7 292.2 316.6 330.3 273.8 293.7 314.8 285.2

32.1 32.4 24.5 32.3 34.1 33.9 29.6 29.0 32.5 25.7

Mean (n = 10) S.D.

299.4 21.0

30.6 3.2

Dosea (mg/(kg bw day))

Serum total genisteinb (␮M)

Serum aglycone genisteinb (␮M)

– – – – – 51 54 49 52 50



51 1.8

1.22 1.30

0.042 0.037

a Doses were estimated from total diet consumed by each dam over the 3 days period from PND7–10, the measured body weight at PND10 and the genistein content in the diet (500 ␮g/g). b Serum genistein concentrations were determined on PND10 (means of three determinations for each genistein-exposed dam, n = 5).

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Table 2 Genistein exposure data and estimates of dosing from suckling Sprague–Dawley pups Litter #

BW gain PND7–10 (g/(day pup))

Milk total genisteina (␮M)

Milk aglycone genisteina (␮M)

Est. doseb (mg/(kg bw day))

Serum total genisteinc (␮M)

Serum aglycone genisteinc (␮M)

1-Control 2-Control 3-Control 4-Control 9-Control 5-Genistein 6-Genistein 7-Genistein 8-Genistein 10-Genistein

1.48 1.46 1.25 1.60 1.53 1.12 1.47 1.21 1.17 1.32



– – – – – 0.44 0.55 0.30 0.40 0.87



0.47 0.21

0.14 0.08

0.51 0.22

0.039 0.011

0.001 0.001

Mean (n = 5) S.D.

Milk genistein concentrations were determined on PND7 (means of two determinations for n = 5 dams); LOD = 0.006 ␮M. Doses were estimated from total body weight gains over the PND7–10 range and the concentration of total genistein in the respective dam’s milk, assuming a 50% conversion factor for milk-derived body weight gain. c Serum genistein concentrations were determined on PND10 (means ± S.D. for n = 8 pups in five genistein-exposed litters except litter #10 where n = 5 pups); LOD = 0.0005 ␮M. a

b

than for pups on control diet dams (1.26 ± 0.20 g/day versus 1.46 ± 0.13 g/day, p = 0.02, Table 2). This observation is consistent with the significant depression of pup body weight gains by genistein that was observed during the multi-generation studies [1]. The litter average daily body weight gain was used to approximate an ingested dose of genistein in each litter by assuming that 50% of the daily body weight gain was produced by milk consumption. The mean estimated dose of genistein delivered to the pups via milk, which was 0.51 ± 0.22 mg/(kg bw day) (Table 2), is approximately 100-fold lower than the mean dose delivered to dams in the feed (Table 1). 4. Discussion This study suggests that the exposures of rat pups to genistein via lactation from dams consuming dietary genistein produces modest circulating levels of both total and aglycone genistein. This low internal exposure apparently results because of the small amount of genistein secreted into the milk (0.12 ppm), relative to either the level in dam food (500 ppm) or blood. The higher concentration of aglycone genistein observed in milk relative to dam blood suggests that preferential secretion occurs, but this does not appear to facilitate overall absorption by the pups. Much as it does in adult rats [28] or humans [29], the distribution of dietary genistein between aglycone and conjugated forms does not affect the total genistein absorbed into the circulation because the bacterial microflora appear to deconjugate efficiently isoflavone glycosides. The fraction of circulating genistein in pups present as the active aglycone species (Tables 1 and 2) is quite similar to that observed in adults and weanlings [7]. This finding is consistent with full development of the relevant Phase II conjugating enzymes in pups. This conclusion contrasts with our previous results, conducted using identical methodology, where a higher fraction of circulating aglycone genistein was observed

in rat fetuses and PND1–2 pups [8], consistent with a generally decreased capacity for Phase II conjugation in rats that increases after birth [30]. Unfortunately, the ontogeny in the rat is not known for UGTs 1A10 and 1A9, the predominant 1A isoforms involved in genistein glucuronidation [31]. It should be noted that the preponderance of conjugated genistein in pup serum does not imply that tissues contain a similar distribution because our measurements of genistein aglycone in adult rats showed much higher percentages of aglycone genistein in tissues (10–100%) relative to blood (1–5%) [7]. The results from this study do not settle the disparate observations in the literature regarding lactational transfer of genistein in rats. The results reported in humans where the level of total genistein transferred to breast milk was less than 10% of the concentrations present in plasma from the mother [19,20] are contrasted with the current study where the corresponding mean ratio was approximately 39%. The measurements of genistein concentrations in dam plasma and milk reported by Lewis et al. [23] and the conclusion that minimal lactation exposure occurs are quite compatible with the present study; however, that study did not quantify genistein in pups. In the study reported by Weber et al. [22], levels of total genistein present in PND3.5 pups were approximately half that of the dams suggesting more efficient transfer than observed in the current study; however, that study did not quantify genistein in milk. Brown and Setchell [32] compared isoflavone levels in blood from dams (0.5 ␮M) fed a soy-containing diet (PMI 5001), PND1 pups (0.35 ␮M), stomach contents from non-suckling PND1 pups which presumably contained amniotic fluid (1.5 ␮M) and PND6 pups (0.3 ␮M). Brown and Setchell [32] concluded that the highest level of developmental exposure to phytoestrogens occurred during the fetal period although their study did not quantify isoflavones in milk. Fritz et al. [21] used a similar comprehensive exposure and analysis protocol to that described here to measure lactational transfer of genistein. Their determinations for total genistein

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in dam serum are consistent with the levels reported here but the values in PND7 pup serum are almost double that of their dams and are much higher than those observed in the present study. Conversely, our values in dam milk were approximately twice as great. Furthermore, the values reported for genistein in pup stomach contents were far higher than in the milk itself [21]. This study shows that only limited lactational transfer of genistein to rat pups occurs under the conditions reported here. This conclusion is compatible with some, but not all literature precedent as described above. This investigation completes the exposure assessment of rats to dietary genistein throughout all life stages (fetal, neonatal, weanling, adult). The higher circulating and, presumably, tissue levels of genistein observed in the fetus relative to the neonate have important implications for the interpretation of dietary multi-generation studies of genistein such as ours. On the one hand, effects attributable to early (preweaning) exposure are more likely to result from fetal exposure because of the higher in utero levels of the active estrogenic aglycone form of genistein. On the other hand, the low exposure to genistein during the sensitive early post-natal period resulting from dietary exposure of the dams needs to be considered when making safety evaluations based on the results of these studies. Acknowledgements The high level technical support provided by Michelle Vanlandingham and Connie Weis and helpful discussions with Dr. John F. Young, all from the NCTR, are gratefully acknowledged. This research was supported in part by Interagency Agreement #224-93-0001 between NCTR/FDA and the National Institute for Environmental Health Sciences/National Toxicology Program. References [1] Delclos KB, Bucci TJ, Lomax LG, Latendresse JR, Warbritton A, Weis CC, et al. Effects of dietary genistein exposure during development on male and female CD (Sprague–Dawley) rats. Reprod Toxicol 2001;15:647–63. [2] Guo TL, White Jr KL, Brown RD, Delclos KB, Newbold RR, Weis CC, et al. Genistein modulates splenic natural killer cell activity, antibodyforming cell response, and phenotypic marker expression in F(0) and F(1) generations of Sprague–Dawley rats. Toxicol Appl Pharmacol 2002;181:219–27. [3] Guo TL, Germolec DR, Musgrove DL, Delclos KB, Newbold RR, Weis CC, et al. Myelotoxicity in genistein-, nonylphenol-, methoxychlor-, vinclozolin- or ethinyl estradiol-exposed F1 generations of Sprague–Dawley rats following developmental and adult exposures. Toxicology 2005;211:207–19. [4] Flynn KM, Ferguson SA, Delclos KB, Newbold RR. Effects of genistein exposure on sexually dimorphic behaviors in rats. Toxicol Sci 2000;55:311–9. [5] Scallet AC, Wofford M, Meredith JC, Allaben WT, Ferguson SA. Dietary exposure to genistein increases vasopressin but does not alter betaendorphin in the rat hypothalamus. Toxicol Sci 2003;72:296–300. [6] Scallet AC, Divine RL, Newbold RR, Delclos KB. Increased volume of the calbindin D28k-labeled sexually dimorphic hypothalamus in genistein and nonylphenol-treated male rats. Toxicol Sci 2004;82:570–6. [7] Chang HC, Churchwell MI, Delclos KB, Newbold RR, Doerge DR. Mass spectrometric determination of genistein tissue distribution in

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