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Division of Biochemical Toxicology, National Center for Toxicological Research, Jefferson, AR 72079 and * Developmental Endocrinology Section, Reproductive Toxicology Group, Laboratory of Toxicology, Environmental Toxicology Program, National Institute of Environmental Health Sciences, Research Triangle Park, NC 27709
3To whom correspondence should be addressed.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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KEY WORDS: • genistein • isoflavones • mass spectrometry • rats • pharmacokinetics
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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for a
review]. In addition, several possible adverse effects have been
suggested, including estrogen agonist activity during critical growth
and developmental periods (Hilakivi-Clarke et al. 1999
,
Hsieh et al. 1998
), antithyroid activity (Divi et al. 1997
) and effects on cognitive function (White, 2000
).
Isoflavones are present in soy mainly as ß,D-glucoside
conjugates; the best evidence suggests that aglycones are liberated in
the gut by the action of microbial ß-glucosidases before absorption
into intestinal cells where glucuronidation presumably occurs before
transfer to the blood (Doerge et al. 2000
,
Sfakianos et al. 1997
). It has been reported that
administration of genistein as either glycosides or the aglycone does
not affect the total absorption, although small differences in peak
blood concentrations were observed (King et al. 1996
).
In this study, genistein aglycone was administered in fortified feed to
Sprague-Dawley rats.
Critical components for assessment of any pharmacologic activity are the distribution and elimination of active compound and metabolites in blood and potential target tissues. This communication describes the development and validation of analytical methodology that was used to determine the pharmacokinetics in blood and distribution of genistein in tissues from rats exposed through continuous dietary intake in a multigeneration test. The strengths of this approach are the defined feed composition and dosing conditions, the rugged, sensitive, accurate and precise analytical determinations derived from use of mass spectrometry and deuterium-labeled genistein as an internal standard, the presentation of method validation data and the application to potential endocrine-responsive target tissues for genistein.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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Genistein was obtained from Toronto Research Chemicals (Toronto, Canada); daidzein, crude glucuronidase/sulfatase from Helix pomatia containing 108 unit/L glucuronidase activity + 5 x 106 U/L sulfatase activity were obtained from Sigma Chemical (St. Louis, MO). Deuterated daidzein (6,3',5'-D3, 95%) and genistein (6,8,3',5'-D4, 95%) were purchased from Cambridge Isotope Laboratories (Andover, MA). All solvents were HPLC grade and Milli-Q water was used throughout.
Rat feeding procedures.
All procedures involving care and handling of rats were reviewed and
approved by the NCTR Laboratory Animal Care and Use Committee. CD
(Sprague-Dawley) rats were from the NCTR colony. The pups usedwere from
parents that had been consuming soy- and alfalfa-free feed
fortified with genistein aglycone at various levels (0, 5, 100 or 500
µg/g feed, which is equivalent to 0, 18.5, 370 or 1852
µmol/kg) continuously since weaning. Male and female
littermates (n = 2 each) from each of six different
litters from each dose group were selected, for a total of 96 rats.
Half of the rats (n = 6 per sex, representing 6
different litters, per dose group) were killed at weaning [postnatal d
(PND)4
21]. After weaning, the remaining 48 pups were fed the same
genistein-supplemented feed received originally by the dam and were
maintained on this food source until the time of killing at PND 140.
The base diet was irradiated 5K96 meal, which contains 13.10 kJ/g,
18–22% protein, 3.8% fiber and 4.6% fat (Purina Mills, St. Louis,
MO). This diet is similar to the standard NIH 31 rat feed
(Knapka 1983
) 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 µg/g) and daidzein (0.48
µg/g) concentrations in this feed were determined
using liquid chromatography-electrospray/tandem mass spectrometry
(LC-ES/MS/MS) analysis after complete hydrolysis of glucoside
conjugates (not shown). The genistein-fortified feed concentrations
were verified using LC-UV (260 nm detection). Complete details
regarding feed consumption and body weights from a related dose
range–finding study will be published separately (Delclos et al.,
unpublished data).
Sample Collection Procedures.
Rats (PND 140) were removed from feed early in the morning (0700 h) randomly with respect to dose group. For pharmacokinetic determinations, ~100-µL portions of blood were removed from the tail vein at 0, 4, 8 and 12 h after removal from food, transferred to Microtainer serum separator tubes (Becton Dickinson, Franklin Lakes, NJ) for preparation of serum by centrifugation after clotting; the serum was stored frozen at -60°C. Rats were given food overnight before killing on the following morning within 3 h after food removal. Blood was removed by cardiac puncture. Tissues (mammary gland, uterus, ovary, testes, prostate, thyroid, liver and brain) were surgically dissected and frozen in liquid nitrogen before storage at -60°C. In the case of weanling rats (PND 21), blood samples were taken immediately after the pups were separated from their dams. Weaning of the pups occurred slightly later in the day than the blood collection for the PND 140 rats and, as was the case for the older rats, the time of last ingestion of genistein-containing feed and/or milk is not known.
Serum genistein analysis.
Rat serum was analyzed for total genistein (i.e., after enzymatic
deconjugation) using isotope dilution LC-ES/MS based on a method
published previously (Holder et al. 1999
). The sample
preparation method reported here substituted automated on-line
solid phase extraction in place of manual liquid-liquid extraction
for analysis of deproteinated serum (acetonitrile precipitation). The
liquid handling system consisted of an autosampler (AS3500, Dionex,
Sunnyvale, CA), an automated switching valve (TPMV, Rheodyne, Cotati,
CA) and an HPLC pump (Dionex GP40). The switching valve allowed the
gradient pump eluent to either load a sample onto the trap column and
then wash, or back flush the trap column contents onto the analytical
column [see Doerge et al. (1999)
for details]. The
sample (0.5 mL) was loaded for 3 min at 1 mL/min with water onto a
reverse-phase trap column (POROS 10-R2, 2.1 x 30 mm,
PerSeptive Biosystems, Framingham, MA); the trap column was then washed
with 7% acetonitrile and 93% of 0.1% aqueous formic acid for 3 min
at 1 mL/min to waste. After the valve was switched, the concentrated
sample zone was backflushed from the trap column onto the analytical
column (Ultracarb ODS3, 2 x 150 mm, 5 µm,
Phenomenex, Torrance, CA) at 0.2 mL/min with 55% of 0.1% formic
acid/45% acetonitrile and eluted into the mass spectrometer. When the
9-min run was finished, a rapid gradient ramp up to 90% acetonitrile
in 2 min increments was used to clean the analytical column. Finally,
the mobile phase was switched back to 45% acetonitrile in a 2-min
gradient step and the analytical column reequilibrated for 5 min. Then
the valve was switched to the initial position to achieve the
following: 1) wash the trap column with 95% aqueous
acetonitrile, 2) equilibrate the trap column with water
and 3) repeat the entire process. It should be noted
that all valve switching was done under computer control from the LC
gradient pump software in a total run time of 32 min. The method was
also adapted to use tandem mass spectrometry in the selected reaction
monitoring mode for the increased sensitivity analysis (~10-fold)
that was required for accurate determination of genistein in serum from
control and 5 µg/g dose groups. Method quantification
limits (s/n = 10) were estimated to be 0.020
µmol/L for LC-ES/MS and 0.005 µmol/L
for MS/MS.
Tissue genistein analysis.
Tissues were thawed, portions excised at random (except brain where one
hemisphere was used), weighed (20–300 mg) and placed in glass tubes.
Citrate buffer (25 mmol/L, pH 5.0) was added (1 mL/100 mg tissue) and
the tissue was homogenized for 30–60 s at room temperature. A portion
of the homogenate containing 20 mg tissue equivalent was transferred to
another glass tube and 800 µL methanol was added
(total volume 1.0 mL). At this point, the internal standard [see
Holder et al. (1999)
for complete details] was added (1
pmol/mg tissue). The resulting suspensions were sonicated for 10 min
and the sample was then mixed with 2 mL citrate buffer. Samples were
processed in duplicate for aglycone determination without enzymatic
deconjugation and for total genistein determination. Deconjugation was
accomplished by adding 100 µg of an enzyme mixture
from Helix pomatia containing 40–60 U of
glucuronidase activity and 1.5–4 U of sulfatase activity
and incubating at 37°C for 60 min as previously described
(Holder et al. 1999
). For fatty tissues such as mammary,
testes, prostate, uterus and ovary, 200 mg NaCl was then added to the
samples and lipids were removed by shaking with three 1.5-mL volumes of
hexane. All samples were centrifuged at 6000 x g for 10 min; then
genistein was purified by solid phase extraction (SPE) using 30-mg
Oasis HLB cartridges (Waters, Milford, MA). The SPE cartridges were
activated by sequentially washing with 1 mL methanol, 1 mL water and
then 1 mL of the citrate buffer. The supernatants were loaded onto the
cartridges, washed with 1 mL water and then the genistein was eluted
with two 1-mL volumes of methanol. The methanolic extracts were reduced
to dryness by using a centrifugal evaporation system (SpeedVac, Savant
Instruments, Farmingdale, NY); the residue was dissolved in 25
µL methanol and 25 µL of water was
added. The samples were mixed and loaded into conical glass inserts for
LC/MS analysis of 45-µL injections as described below.
Some tissues (mammary, uterus, ovary, thyroid) from control rats contained small peaks in the LC-ES/MS chromatograms that could have arisen from either artifacts or the small amount of genistein consumed in the control diet. For this reason, control and low dose samples for these tissues were also analyzed using LC-ES/MS/MS to add specificity to the detection method. Reduced responses were observed using MS/MS, suggesting that the ES/MS peaks were due in part to tissue matrix interferences. It is likely that residual values, determined using MS/MS, did result from the small amount of genistein present in the control rat feed and no correction was made to the reported values. No correction was made for the contribution to total genistein due to the content of residual blood in tissues because such information was not available about all tissues. Aglycone measurements were not made in control and other tissues in which only small amounts of total genistein were likely. The limit of detection (LOD, s/n = 3) for genistein varied depending on the tissue matrix and its level of background responses, but was in the range of 0.04–0.09 pmol/mg for LC-ES/MS and 0.01–0.03 pmol/mg for MS/MS. The low genistein levels measured in brain tissue using LC-ES/MS were confirmed by repeating these analyses using MS/MS.
The analytical LC separation for tissue genistein was performed as described for the serum analysis but without the column switching procedures. System quality control measures included regular injections of blanks and standards containing a mixture of labeled and unlabeled genistein standards throughout each sample set to provide a check of instrument responses and the recovery of the internal standard in tissue samples.
Mass spectrometry.
Either a Platform II single quadrupole or a Quattro LC triple
quadrupole mass spectrometer (Micromass, Manchester, U.K.) equipped
with an ES interface was used with an ion source temperature of
150°C. Positive ions were acquired in selected ion monitoring mode
for ES/MS (dwell time = 0.3 s, span = 0.02 Da and
interchannel delay time = 0.03 s) for the protonated molecule
(M + H)+ ions for genistein (m/z 271) and
genistein-d4 (m/z 275), which were monitored at a
sampling cone-skimmer potential of 30 V. Alternatively, for MS/MS
measurements, a collision energy of 25 eV was used for the (M +
H)+ transitions used to monitor unlabeled and deuterated
geinstein (m/z 271 
215 and 275 219,
respectively) with a dwell time of 0.3 s and a sampling
cone-skimmer potential of 50 V. The validation of the isotope
dilution technique for analysis of genistein and characterization of
the deuterated genistein was reported previously in detail
(Holder et al. 1999
).
Statistical analysis.
Serum levels of genistein from PND 140 rats were analyzed by two-way ANOVA with sex and dose as categorical variables. Comparisons between genistein levels in males and females at each dose level were made using a t test with a Bonferroni correction for multiple comparisons. Pharmacokinetic values [half-life and area under the concentration curve (AUC)] in males and females were compared using t tests. Tissue levels of aglycone and total genistein were analyzed separately by sex using one-way ANOVA. Graphical examination of the data as well as testing for homogeneity of variances using the Brown-Forsythe modification of Levene’s test (Statistica v5.5, StatSoft, Tulsa, OK) indicated that the data for the majority of tissues violated the ANOVA assumption of homogeneous variances. Analyses of tissue levels were therefore conducted on log-transformed data, which had homogeneous variances. Before log transformation, values below the limit of detection were assigned a value of 0.5 times the limit of detection. When the ANOVA showed a significant treatment effect, pairwise comparisons of each dose group against the control were made with Dunnett’s test. In cases in which the aglycone content of the control group was not determined, the control values of total genistein were used in Dunnett’s test. All statistical tests were considered significant at the P < 0.05 level. The exception was the Brown-Forsythe test, for which the significance level was P < 0.01.
Pharmacokinetic determinations.
Plots of serum genistein vs. time were prepared for individual rats and
appeared to follow a single exponential decline. The first-order
elimination rate constant and AUC (zero to infinity) were determined
graphically for individual rats (Ritschel 1986
). At the
8-h time point, the tail vein bleeding for three females provided
insufficient sample volumes; therefore, aliquots of serum from these
rats were pooled to provide an additional sample and this value was
used for the missing values. Similarly, no samples were collected from
two male rats at the 8-h time point; thus the average value from the
other four males in the group was used for pharmacokinetic
determinations of these two individuals. The kinetic parameters derived
from using these substitutions were evaluated against removal of the
time points and were judged to improve or maintain the overall data
fit.
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RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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An on-line SPE-LC/MS method was used for the automated analysis of
serum genistein. Figure 1
shows representative chromatograms for control and incurred serum, and
the respective labeled and unlabeled standards. This incremental
improvement of a previous method was implemented to increase the level
of automation. The methods were validated by analyzing control serum,
control serum spiked with genistein and incurred serum samples
containing 1–6 µmol/L genistein on each day samples were
analyzed (5 total). Recovery of spiked genistein (1
µmol/L) was in the range of 78–89%, based on the signal
of the deuterated genistein compared with an external standard analyzed
in parallel. The intra-assay precision ranged between 1 and 5%
relative standard deviation (RSD) for analysis of replicate incurred
serum samples on each day of sample analysis (n = 5).
Interassay precision and accuracy were evaluated using control serum
spiked with 2.5 µmol/L genistein. Initially, the value
determined was 2.20 ± 0.12 µmol/L (5% RSD,
n = 4); on another day, the value was 2.23 ± 0.13
µmol/L (6% RSD, n = 4). The level of
method performance was similar to that reported previously
(Holder et al. 1999
). Although the method required
considerably less sample preparation time than off-line
liquid-liquid extraction, it also resulted in the introduction of
additional serum components onto the LC column. Therefore, it was
necessary to incorporate a short gradient cleanup step at the end of
each chromatographic run to maintain satisfactory column performance.
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). The serum from control rats
contained small amounts of total genistein (<0.01 µmol/L,
see Fig. 1
, middle panel), presumably from the low level of
isoflavones present in the "soy-free" feed. For this reason, the
LOD in serum from genistein-fed rats was set at 0.01
µmol/L. Table 1
shows the serum levels obtained immediately after food was removed from
groups of rats consuming genistein. These values, which approximate the
maximal blood concentrations because of uncertainty about the time of
last feeding, increased monotonically with dose in both male and female
adult rats and were similar to those published previously from a
related study (Holder et al. 1999
). Analysis of the data
by two-way ANOVA indicated significant effects of sex (P
= 0.017) and dose (P < 0.001) as well as a dose
x sex interaction (P = 0.017). No significant
differences were observed when the serum genistein concentrations for
males and females at individual dose levels were compared.
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). It should be noted that these pups were probably exposed to genistein
in maternal milk and feed and that blood was taken immediately after
removal of the pup from the dam. The serum genistein concentrations of
weanling rats also increased monotonically with dose, although no sex
difference was observed. The preponderance of circulating genistein in
weanling blood was present in conjugated forms as observed previously
in adult rats (95–99%, Holder et al. 1999
). These
serum levels for total genistein are similar to those reported
previously in plasma of 7- and 21-d-old female Sprague-Dawley rats,
although in that study, the aglycone content was reported to be 14 and
7%, respectively (Fritz et al. 1998
).
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The pharmacokinetics of genistein elimination were evaluated in PND 140
rats consuming the 500 µg/g genistein diet by collecting
blood sequentially beginning immediately after removal of food. This
zero time point is assumed to best approximate the peak serum genistein
concentrations in this study. The combined data for male and female rat
groups are plotted in Figure 2
and were consistent with a single phase elimination process.
Pharmacokinetic parameters for males and females, computed using
individual rat results, were as follows: half-times, 2.97 ± 0.14 h and 4.26 ± 0.29 h, respectively (P
< 0.004); AUC, 22.3 ± 1.2 and 45.6 ± 3.1,
respectively (P < 0.001). The rapid elimination of
genistein is similar to that reported previously in male Wistar rats
(8.8 h, King et al. 1996
) and humans (8.4 h,
Watanabe et al. 1998
; 5.7 h, King and Bursill 1998
; 7.9 h, Setchell1998
) and
urinary elimination in humans (4–7 h, Lu and Anderson 1998
).
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), whereas Lu and Anderson (1998)
found a longer excretion half-life for genistein and
other isoflavones in women compared with men after a single ingestion
of soy milk delivering 80–210 mg of each isoflavone. Tissue genistein analysis.
The organs analyzed in this study were chosen because of their
endocrine-responsive nature and because previous studies (Delclos
et al., unpublished data) indicated that genistein produced effects
detectable by microscopic evaluation. Total tissue genistein and the
aglycone fraction were determined because most in vitro studies
reported to date evaluated effects with only the aglycone. However, the
limited reports of isoflavone conjugate activity suggest that the
aglycone is the more active form (Divi et al., 1997
,
Zhang et al. 1999
). Method performance was evaluated by
measuring the precision and accuracy of replicate determinations
(n 
3) from control tissues spiked at 1 pmol/mg with
labeled and unlabeled genistein for every tissue examined (not shown).
The precision of determination was in the range of 1–9% RSD, and the
accuracy of determination was 83–115% of the spike value. Recovery of
genistein from tissues, measured against a parallel external standard,
ranged from 40 to 78%. A representative set of chromatograms is shown
in Figure 3
.
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and
4
show the results from analysis of genistein in selected tissues from
male and female rats. Dose-related increases in genistein
concentration were observed in all organs examined. Statistical
analysis (ANOVA and Dunnett’s test) indicated that all total genistein
and most aglycone determinations in the rats given the 100 and 500
µg/g doses were significantly different from the control
values. In all tissues examined, the fraction of total genistein
present as aglycone (10–100%) exceeded the fraction in blood
(1–5%). Notable were the mammary gland, uterus, ovary, prostate,
brain and the female liver in which at least half was present as
aglycone.
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The genistein concentration in female mammary gland (Table 4)
was
similar to that reported previously in 7- and 21-d-old female
Sprague-Dawley rats (i.e., the ratio of total mammary
genistein/blood genistein; Fritz et al. 1998
). However,
the total mammary genistein observed previously was lower (250
µg/g dietary genistein led to ~0.4 pmol/mg) and the
aglycone fraction was higher (72–83%) (Fritz et al. 1998
). The total genistein concentration and the percentage of
aglycone found in whole prostate (Table 3)
are similar to but somewhat
higher than values reported previously for dorsolateral prostate from
Lobund-Wistar rats fed genistein-containing diets (Dalu et al. 1998
).
These results extend to more organs, the previous observations of
genistein tissue distribution in mammary and prostate glands
(Dalu et al. 1998
, Fritz et al. 1998
) and
suggest that the small amounts of nonpolar aglycone in blood can
partition into many lipophilic tissues and accumulate. A finding that
supports this conclusion is the relative genistein content in male vs.
female mammary gland. The female gland, presumably because of its
higher lipid content, accumulated more genistein, in both aglycone and
conjugated forms. Of course, factors other than tissue concentration of
genistein may be important determinants of the tissue response. In the
mammary glands of 50-d-old rats, males showed responses at lower doses
than did females (Delclos et al. 1999
). Although it is
possible that the relative tissue levels in males and females may
differ from those reported here in the younger rats, it seems more
likely that the responsiveness of the mammary glands to genistein
differs between the sexes.
Brain genistein.
The limited accumulation of genistein in brain tissue is striking by
comparison with all other tissues examined and may reflect poor
penetration of isoflavones into the central nervous system (CNS) of
adult rats. In the case of other compounds that are highly conjugated
in the circulation (e.g., morphine), glucuronides have been shown to
penetrate the blood-brain barrier much less than the parent
compound (Bickel et al. 1996
, Wu et al. 1997
). A separate analysis of a single brain from a pregnant
female rat dosed orally by gavage with genistein revealed a slightly
higher level of genistein in the brain (0.27 pmol/mg) 2 h after
dosing than that shown in Table 4
, despite comparable blood genistein
concentrations. This result may reflect differences in the rate of
compound administration (i.e., bolus vs. continuous consumption),
enhanced elimination of genistein from the brain relative to the
circulation or possible differences in genistein metabolism in the
pregnant female (e.g., lower glucuronidation than the nonpregnant
adult). The finding of low genistein values in the adult brain is based
on analysis of individual brain hemispheres; therefore, no conclusions
about penetration into or localization in specific sites in the CNS can
be inferred, especially those structures outside the blood-brain
barrier. A previous study observed, but did not quantify genistein and
a metabolite, p-ethylphenol, in the cerebrospinal fluid of
rats (Setchell 1998
).
The apparent limited penetration into the adult brain would in no way
preclude the ability of genistein to produce by indirect mechanisms the
alterations in brain function that have been reported in genistein- or
soy-exposed rats (e.g., Faber and Hughes 1993
,
Pan et al. 1999
, Taylor et al. 1999
,
Trieu and Uckun 1999
). First, some effects, such as
those on sexually dimorphic nuclei in the brain (Faber and Hughes 1993
, Taylor et al. 1999
), are determined
by exposures early in life, before the development of the
blood-brain barrier. Preliminary results obtained from rat pups of
dams treated with a single oral dose of genistein aglycone and killed
2 h later indicated that genistein did cross the placenta into the
pups’ circulation and that measurable levels of genistein were found
in the fetal brains (not shown). The results of Fritz et al. (1998)
also indicated that newborn pups are exposed to
genistein and a relatively high percentage of aglycone, via maternal
milk. Alternatively, isoflavones have been reported to alter sex
steroid hormone levels and metabolism (Xu et al. 1998
and references cited therein); these or other peripheral effects may
have indirect effects on CNS function.
Liver genistein.
A final intriguing observation from this study is the 5- to 11-fold
excess in total genistein concentration and a more than 100% increase
in the aglycone fraction observed in female vs. male liver. As
discussed above in light of sex differences in blood genistein, the
basis for the difference in liver genistein concentration is not clear.
One possibility would be differences in perfusion of the liver of the
females secondary to an increased liver volume. However, the liver
weights of rats treated in a parallel study did not show significant
treatment-related effects. A previous shorter study in which pups
were exposed to dietary genistein, up to 1250 µg/g through
PND 50, found minimally increased liver to body weight ratio (~9%)
only at the high dose and only in males (Delclos et al., unpublished).
A previous report of comparable receptor
3H-estradiol binding activity in livers of male
and female rats (Lax et al. 1983
) suggested that total
estrogen receptor (ER) content is not the cause of the large sex
difference in liver genistein concentration. A recent study reported
that ER
, but not ERß mRNA was expressed in male rat liver but
female liver was not examined (Kuiper et al. 1997
).
Another study reported that the livers of male and female mice
expressed large amounts of ER, but only small amounts of ERß
(Couse et al. 1997
). Despite the reported ninefold
higher affinity of ERß for genistein relative to ER (Kuiper et al. 1998
), small differences in receptor subtype numbers are
not likely to affect total binding of genistein. The broad specificity
of human IA class UDP glucuronosyl transferases and sulfotransferases
for genistein was reported previously (Doerge et al. 2000
); therefore, the similar presence of these enzymes in rat
liver suggests that sex differences in expression cannot explain the
observed difference in aglycone content.
On the basis of the serum concentrations of total genistein (Table 1)
,
adult rats fed the control and low dose (5 µg/g) diets are
similar to human adults consuming a typical Western diet (<0.1
µmol/L); rats fed the 100 µg/g genistein diet
are similar to human adults consuming a typical Asian diet (0.1–1.2
µmol/L, Adlercreutz et al. 1994) or soy
nutritional supplements (0.5–0.9 µmol/L, Doerge et al. 2000
); and rats fed the 500 µg/g genistein
diet are similar to infants consuming soy formulas (2–7
µmol/L, Setchell et al. 1997
). The higher
levels of genistein aglycone observed in many endocrine-responsive
rat tissues relative to the blood suggest that accumulation is typical
and that similar tissue levels can occur in human populations.
|
ACKNOWLEDGMENTS |
|---|
|
FOOTNOTES |
|---|
2 H.C.C. acknowledges support of a fellowship from the Oak Ridge Institute for Science and Education, administered through an interagency agreement between the U.S. Department of Energy and the U.S. Food and Drug Administration.
4 Abbreviations used: AUC, area under the concentration-time curve; CNS, central nervous system; ER, estrogen receptor; ES, electrospray; LC, liquid chromatography; LOD, limit of detection (s/n = 3); (M+H)+, protonated molecule; MS, mass spectrometry; PND, postnatal day; RSD, relative standard deviation; SPE, solid phase extraction.
Manuscript received January 19, 2000. Initial review completed February 15, 2000. Revision accepted March 23, 2000.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
|---|
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4. Delclos K. B., Lomax L. G., Weis C. C., Holder C. C., Doerge D. R., Newbold R. R. Effects of dietary genistein exposure during development on Sprague-Dawley rats. Toxicol. Sci. 1999;48S:47
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