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Nutrition and Cancer
Brown Kelp Modulates Endocrine Hormones in Female Sprague-Dawley
Rats and in Human Luteinized Granulosa Cells1
Christine F. Skibola,*2 John D. Curry,*3 Catherine VandeVoort,† Alan Conley,** andMartyn T. Smith* *School of Public Health, University of California, Berkeley, California; and †California National PrimateResearch Center and **Department of Population Health and Reproduction, School of Veterinary Medicine,University of California, Davis, California Epidemiological studies suggest that populations consuming typical Asian diets have a lower incidence of hormone-dependent cancers than populations consuming Western diets. These dietary differenceshave been mainly attributed to higher soy intakes among Asians. However, studies from our laboratory suggest thatthe anti-estrogenic effects of dietary kelp also may contribute to these reduced cancer rates. As a follow-up toprevious findings of endocrine modulation related to kelp ingestion in a pilot study of premenopausal women, we investigated the endocrine modulating effects of kelp (Fucus vesiculosus) in female rats and human luteinizedgranulosa cells (hLGC). Kelp administration lengthened the rat estrous cycle from 4.3 Ϯ 0.96 to 5.4 Ϯ 1.7 d at 175mg ⅐ kgϪ1 body wt ⅐ dϪ1 (P ϭ 0.05) and to 5.9 Ϯ 1.9 d at 350 mg ⅐ kgϪ1 ⅐ dϪ1 (P ϭ 0.002) and also led to a 100%increase in the length of diestrus (P ϭ 0.02). Following 175 mg ⅐ kgϪ1 ⅐ dϪ1 treatment for 2 wk, serum 17␤-estradiol levels were reduced from 48.9 Ϯ 4.5 to 40.2 Ϯ 3.2 ng/L (P ϭ 0.13). After 4 wk, 17␤-estradiol levels were reducedto 36.7 Ϯ 2.2 ng/L (P ϭ 0.02). In hLGC, 25, 50, and 75 ␮mol/L treatment reduced 17␤-estradiol levels from 4732 Ϯ 591 to 3632 Ϯ 758, 3313 Ϯ 373, and 3060 Ϯ 538 ng/L, respectively. Kelp treatment also led to modest elevationsin hLGC culture progesterone levels. Kelp extract inhibited the binding of estradiol to estrogen receptor ␣ and ␤ andthat of progesterone to the progesterone receptor, with IC values of 42.4, 31.8, and 40.7 ␮mol/L, respectively.
These data show endocrine modulating effects of kelp at relevant doses and suggest that dietary kelp maycontribute to the lower incidence of hormone-dependent cancers among the Japanese.
KEY WORDS: ● Fucus vesiculosus ● rat ● seaweed ● breast cancer ● estrogen The rise in estrogen-dependent cancers in the United with abnormal menstrual cycling histories (10). In the present States and our limited success with their prevention and study, we have further investigated the endocrine modulating treatment have spurred growing interest in the dietary habits effects of F. vesiculosus on sex hormone levels and cycling of the Japanese, who have one of the lowest rates of breast, patterns in rats and in a human model.
endometrial, and ovarian cancers in the world (1,2). Studies Previous studies show an inverse relation between men- show that Japanese women have longer menstrual cycles and strual cycle length and risk of breast (11), ovarian (12), and lower serum estradiol levels than their Western counterparts endometrial (13) cancers. Menstrual cycle length and age of (3–5), factors that may contribute to their low risk of estrogen- onset of menarche and menopause may serve as surrogate dependent cancers. To date, these low rates have been partly measures of endogenous estradiol and progesterone exposure attributed to the soy-rich diets inherent among Asian popu- [reviewed in (14)]. Women with shorter cycles experience a lations (6 – 8). However, another contributory factor may be greater total number of menstrual cycles during the course of their high intake of seaweed, as previously hypothesized by their reproductive lifetimes than those with longer cycling Teas et al. (9). In a human pilot study, we demonstrated that patterns. Hence, these women will spend more time overall in intake of the brown kelp seaweed, Fucus vesiculosus (bladder- the follicular and luteal phases of the cycle where estrogen and wrack), significantly increased the total number of days of the progesterone levels and endometrial and breast cell prolifera- menstrual cycle, reduced circulating 17␤-estradiol levels, and tion rates are at their highest. Positive associations also have elevated serum progesterone levels in premenopausal women been reported among breast, endometria, and ovarian cancersand obesity and alcohol intake, factors that promote persistent estrogenic stimulation and hormone imbalances (15–17).
Supported by the National Foundation for Cancer Research and NIH Grant These studies suggest that exposure to estrogens and an im- 2 To whom correspondence should be addressed.
balance in the estrogen/progesterone ratio may be the most critical determinants in risk of estrogen-dependent diseases.
Present Address: Division of Molecular and Cellular Biology, University of Inhibition of estrogen via the estrogen receptor or aromatase 0022-3166/05 $8.00 2005 American Society for Nutritional Sciences.
Manuscript received 14 September 2004. Initial review completed 27 October 2004. Revision accepted 22 November 2004.
ENDOCRINE MODULATION BY Fucus vesiculosus blockade is a current strategy for prevention in high-risk Kelp dose finding experiment (Expt. 1). Normally cycling rats
individuals and in the treatment of some estrogen-dependent were randomly divided into 3 groups of 8: a vehicle control, a low diseases. Furthermore, the identification of dietary compo- dose (175 mg ⅐ kgϪ1 ⅐ dϪ1), and a high dose (350 mg ⅐ kgϪ1 ⅐ dϪ1) nents that exert chemoprotective effects by suppressing en- group. Powdered F. vesiculosus was measured and applied in the dogenous estrogen production may provide another means to morning daily to a 2-g fresh apple wedge used as a vehicle. There wasa dual advantage to using an apple vehicle in this study: (1) it reduce the incidence of breast, endometrial, and ovarian can- eliminated stress associated with gavage; (2) rats eagerly ate the apple and the kelp in this manner, making it easy to monitor and ensure Rat and primary human luteinized granulosa cell (hLGC)4 complete deliverance of the kelp. Vaginal smears were obtained and models have been utilized to examine chemical endocrine daily logs were maintained to monitor estrous cycling. After ϳ4 wk, disruptor effects on disease risk. The Sprague-Dawley female all rats were removed from treatment. No adverse effects were ob- rat has been used as a model to investigate the effects of served during the course of the experiment.
endocrine modulation on mammary, ovarian, and endometrial Time course experiment (Expt. 2). For serum hormone studies
carcinogenesis (18 –20). Chemical endocrine disruptors are and to determine whether dosing over time altered hormone levels, commonly tested using the rat model due to similarities in 1-mL blood draws were taken from the tail veins of 19 normally ovarian hormone responsiveness between humans and rats cycling rats during the morning of proestrous (determined by vaginal Downloaded from (21,22). In both the rat estrous cycle and the human menstrual cytology). Immediately following the blood draw, rats were given 175mg ⅐ kgϪ1 ⅐ dϪ1 kelp. At 2- and 4-wk intervals, blood was redrawn cycle, estradiol levels peak during proestrus and the follicular during the morning of proestrus. Blood samples were allowed to clot phase and progesterone levels peak during diestrus and the at room temperature and were centrifuged for 10 min at 2000 ϫ g.
luteal phase, respectively. Primary hLGC cultures simulate Serums were aspirated and frozen at Ϫ20°C until further analysis.
granulosa lutein cells of the corpus luteum in vivo because High dose experiment (Expt. 3). To determine whether high
they support the timely and dynamic secretion of estradiol and dose kelp treatments would exert anti-estrogenic and/or progesta- jn.nutrition.org progesterone in patterns that mimic serum hormone levels genic effects in rats with high circulating estradiol levels, 8 rats were during the luteal phase of the menstrual cycle. Thus hLGC chosen whose estradiol levels were approximately Ն50 ␮g/L. Rats cultures have been utilized successfully in the study of chem- were dosed 350 mg ⅐ kgϪ1 ⅐ dϪ1after a baseline blood draw was taken ical endocrine disruption, such as the effects of dioxin on during the morning of proestrus. Following 1 wk of treatment, an ovarian estrogen and progesterone synthesis (22).
additional blood draw was taken during the morning of proestrus.
Our objectives in the present study were to build upon our Animal hormone assays. Blood serum progesterone was assayed
in triplicate using an ELISA kit (Product No. DSL-10 – 6800) from previous findings using these models by 1) examining whether Diagnostic Systems Laboratories according to the manufacturer’s di- dietary administration of F. vesiculosus disrupts normal estrous rections. 17␤-Estradiol was assayed in duplicate by a radioimmune cycling and sex steroid secretion in Sprague-Dawley rats and antibody assay according to the method described previously (25).
2) treating hLGC cultures with a F. vesiculosus extract to study Crude seaweed extractions. Dried, powdered F. vesiculosus (50
differences in endocrine responses in granulosa cells. To fur- g) was placed in a 1:1 solution of distilled water and 100% ethanol, ther investigate possible mechanisms of action of the F. ve- covered, and stirred for 24 h at room temperature. The extract was siculosus extract in estrogen and progesterone responses, we centrifuged for 10 min at 4500 ϫ g, sterile filtered, evaporated to evaluated its binding affinity to estrogen receptor (ER)␣, ER␤, dryness using a rotary evaporator, and resolubilized to the desired and progesterone receptor (PR)-B and its potential to inhibit concentrations in 50% ethanol. The estimated molecular weight of aromatase activity in hLGC cultures.
the crude seaweed extract was 300 g/mol, a value commonly used inthe pharmaceutical industry for testing bioactivity of unknown plantcompounds.
MATERIALS AND METHODS
hLGC culture and treatment.
Rats and estrous cycle monitoring.
from 8 women undergoing assisted reproduction treatment at a fer- Sprague-Dawley rats (Charles River Canada) weighing 200 –250 g tility clinic. Cells were prepared, plated, and cultured as previously were individually housed in wire cages. They were allowed ad libitum described for each patient (26). Briefly, cells were plated in minimum access to a standard laboratory diet (AIN-76) (23) and water. Fol- essential medium (MEM) supplemented with 0.1 IU/mL human lowing a brief 2-wk adjustment period, rats underwent daily vaginal chorionic gonadotropin, antibiotics, and 5% fetal calf serum at 37°C cytology monitoring to determine normal estrous cycling using stag- in an atmosphere of 5% CO in air. After 48 h, cells were treated on ing criteria described by Everett (24). Rats were required to have at consecutive days for 9 d with ethanol (vehicle control) or 25, 50, or least 2 normal, consecutive estrous cycles prior to experimentation. A 75 mmol/L kelp extract. Samples of medium from final day of treat- normal estrous cycle was defined as a 3- to 5-d cycle. A complete ment were assayed for 17␤-estradiol and progesterone.
estrous cycle was defined as the day of estrus to the day before the Granulosa cell hormone assays. Estradiol and progesterone mea-
subsequent estrus. Weights of rats were monitored weekly throughout surements were performed using commercially available RIA kits (Diagnostic Products) as previously reported (27).
Source and dosing of F. vesiculosus. Dried, powdered F. vesicu-
Estrogen and progesterone receptor binding assays. Affinity of
losus was obtained from Maine Coast Sea Vegetables. The kelp was the kelp extract to ER␣, ER␤, and PR-B was determined by radio- harvested from the Gulf of Maine and from the coastal waters of New metric competitive binding assays as previously described (28,29) by Brunswick and Nova Scotia during the late summer months. Process- an outside laboratory (MDS Panlabs). Briefly, dried kelp extract in 3 ing entailed sun drying the entire plant less the holdfast (root sys- dilutions (0.5, 5, and 50 ␮mol/L final concentration) were resolubi- tem). The dried seaweed was then milled into a fine powder.
lized in dimethyl sulfoxide, combined with ER␣ or ER␤ and 0.5 Dosage levels were chosen to fall within the range of effective nmol/L estradiol, and mixed for 2 h at 25°C. Nonspecific binding was doses in our previous human studies and in a traditional Asian diet, estimated in the presence of 1 ␮mol/L diethylstilbesterol. To test taking into account that higher doses are often needed in rats to PR-B binding, kelp extracts were incubated for 2 h with PR-B and 1.4 produce effects comparable to those seen in humans due to the nmol/L radiolabeled progesterone at 4°C. Nonspecific binding was increased rate of metabolism of the rat. Ethical approval was obtained estimated in the presence of 1 ␮mol/L progesterone. All determina- for the study and all of the studies were conducted in full compliance tions were carried out in triplicate and data are means Ϯ SEM.
with the U.C. Berkeley Animal Care and Use Committee guidelines.
Aromatase activity measured using a tritiated water assay.
Aromatase activity was estimated by measuring the incorporation oftritium from androstenedione into 3H O as previously described 4 Abbreviations used: ER, estrogen receptor; hLGC, human luteinized gran- (30,31). Incubations of hLGCs in 500 ␮L MEM with 300 nmol/L ulosa cell; MEM, minimum essential medium; PR, progesterone receptor.
androstenedione (10% labeled, 90% radio inert, Steraloids) were carried out at 37°C for 2 h in the presence or absence of the kelpextract (10, 50, and 100 ␮mol/L).
Statistical analyses. In Animal Expt. 1, differences between the
means of the 3 groups were evaluated by two-way ANOVA usingProc Mixed in SAS and values are means Ϯ SD. Dunnett’s pairwise comparison procedure was used to evaluate the pairwise differencesbetween treatments and the control group. For all other experiments,statistical analyses were performed by paired t tests (2-sided) with acommercially available statistical software package (Statsoft) andresults were considered significant for P Ͻ 0.05. Values are means a nonlinear, least squares regression analysis using Data AnalysisToolbox (MDL Information Systems).
Animal studies
Kelp dose finding experiment (Animal Expt. 1).
estrous cycle was evaluated daily for ϳ30 d in 24 female Sprague-Dawley rats. Kelp administration led to a profound,dose-dependent increase in the length of the estrous cycle in rats fed 175 and 350 mg ⅐ kgϪ1 ⅐ dϪ1 kelp (P ϭ 0.004). In the 17␤-Estradiol levels in rats following 1 wk of 350 controls, the mean number of days of the estrous cycle was 4.3 mg ⅐ kgϪ1 ⅐ dϪ1 kelp administration. The box plots indicate that in rats Ϯ 0.96 compared to 5.4 Ϯ 1.7 in the 175 mg ⅐ kgϪ1 ⅐ dϪ1 dose with high circulating serum 17␤-estradiol levels median serum 17␤- group (P ϭ 0.05) and 5.9 Ϯ 1.9 d in the 350 mg ⅐ kgϪ1 ⅐ dϪ1 estradiol levels significantly decreased from 68.6 to 42.8 ng/L (P dose group (P ϭ 0.002). Furthermore, kelp treatment led to an overall 100% increase in the mean length of the diestrus phaseof the estrous cycle (P ϭ 0.02). Specifically, the mean number of days in diestrus was 0.97 Ϯ 0.22 among the controls com- extracts compete for and bind to ER␣, ER␤, and PR-B, with a pared to 1.4 Ϯ 0.54 for the 175 mg ⅐ kgϪ1 ⅐ dϪ1 dose group and 2.1 Ϯ 0.88 for the 350 mg ⅐ kgϪ1 ⅐ dϪ1 dose group (P ϭ 0.02).
Aromatase activity following treatment of hLGCs with the Treatment had no significant effect on the number of days in kelp extract did not differ (data not shown).
estrus, proestrus, or metestrus during the mean estrous cycle.
Total number of days monitored was 28.6 Ϯ 3.1, 30.5 Ϯ 3.6, DISCUSSION
and 31.9 Ϯ 3.6 for the 0, 175, and 350 mg ⅐ kgϪ1 ⅐ dϪ1 groups, Here we report additional evidence of the anti-estrogenic Following Expt. 1, 5 rats stopped normal estrous cycling and bioactivity of dietary F. vesiculosus by demonstrating its effects were excluded from the remainder of experiments. One re- on rat estrous cycling patterns and serum hormone levels and on estradiol production in treated hLGC cultures. Specifically, Effects on serum estradiol and progesterone levels (Ani-
dietary kelp resulted in an overall 37% increase in the length mal Expt. 2). Following the 175 mg ⅐ kgϪ1 ⅐ dϪ1 treatment
of the rat estrous cycle in a dose-dependent manner and led to for 2 wk, mean serum 17␤-estradiol levels were reduced from a prolonged diestrus phase of the cycle in the 350 48.9 Ϯ 4.5 to 40.2 Ϯ 3.2 ng/L (P ϭ 0.13) and after 4 wk at the mg ⅐ kgϪ1 ⅐ dϪ1 group. Kelp administration also exerted a tem- same dose levels were significantly reduced 25% from baseline pering effect on estrogen production in rats, which led to to 36.7 Ϯ 2.2 ng/L (P ϭ 0.02), suggesting an effect of dosing 18 –33% reductions in circulating 17␤-estradiol levels. These over time. Serum progesterone levels between controls and the findings are consistent with the observed increase in menstrual cycle length and decrease in serum estradiol levels in women High dose experiment (Animal Expt. 3).
following kelp administration (10). Moreover, the doses used with high circulating serum 17␤-estradiol levels following 1 in this study are physiologically relevant doses and are consis- wk kelp administration (350 mg ⅐ kgϪ1 ⅐ dϪ1), median serum tent with the range of intakes of 3–13 g/d estimated in Japa- 17␤-estradiol levels decreased by 38% (P ϭ 0.02) (Fig. 1).
nese populations (32). Previous studies investigating the role The range in reduction of serum 17␤-estradiol levels in 6 rats of dietary soy or genistein on the rat estrous cycle showed was 25–58%, whereas 2 rats did not respond to kelp at all.
either no effects (33) or only a modest 10% increase in cycle Progesterone levels were not significantly affected following length (34), suggesting that kelp may exert a greater effect in increasing cycle length than soy intake.
Effects of kelp treatment on17␤-estradiol and progester-
The anti-estrogenic bioactivity of F. vesiculosus was further one levels in human granulosa cells. In hLGC cultures, the
demonstrated in an hLGC bioassay where dosing with kelp 50 and 75 ␮mol/L doses significantly reduced 17␤-estradiol extract led to 23–35% reductions in 17␤-estradiol levels in cell levels by 30 and 35%, respectively (Table 1). Kelp treatment
cultures. This would suggest that the extract might act by also led to modest elevations in progesterone in hLGC me- either inhibiting estradiol production or enhancing its meta- dium; however, only the 50 ␮mol/L dose was increased (P bolic breakdown. Competitive inhibition, altered expression, or posttranslational modification of any one of a number of Radioligand binding assay.
cytochrome P450 enzymes involved in steroidogenesis (includ- binding assays, the kelp extract exerted inhibitory effects on ing cholesterol transport) or in 17␤-estradiol metabolism the binding of estradiol to ER␣, ER␤, and progesterone to could affect estradiol levels (22,35). However, we found no PR-B (Table 2). These data demonstrate that F. vesiculosus
inhibitory effects of the kelp extract on aromatase activity, ENDOCRINE MODULATION BY Fucus vesiculosus polysaccharides, and the fucosterols found in several brownalgae species. Polyphenols constitute ϳ15% of F. vesiculosus, 17␤-Estradiol and progesterone levels in medium of hLGC with 25% of this fraction consisting of high-molecular-weight treated with 0, 25, 50, or 75 ␮mol/L kelp extract1 polymers. Phlorotannins, oligomers, and polymers of phloro-glycinol (1,3,5-trihydroxybenzene) are the largest polyphe- nolic group found in F. vesiculosus and other brown algae.
These compounds have been shown to exert bactericidal ac- tivity (44), reactive oxygen species inhibition (45), and in- hibitory effects on human immunodeficiency virus type 1 reverse transcriptase and protease (46). The sulfated polysac- charides, known as fucoidans, are also found in brown sea- weeds. Anti-viral (47), anti-proliferative (48), anti-angio-genic, and anti-tumor (49) properties of fucoidans have 1 Values are means Ϯ SEM, n ϭ 4.
recently been described. Because of this complex chemical 2 Based on t tests between the control and the intervention dose.
makeup, the precise nature of the compound(s) responsible for Downloaded from the endocrine-modulating effects described in this study will which was considered a potential molecular target due to the be difficult to elucidate but efforts are underway to isolate fact that a number of plant compounds exert highly specific inhibitory activity against this enzyme (36 –38). Further stud- Our data suggest that brown seaweed intake may contribute ies of the mechanism by which kelp extracts inhibit estradiol to the lower hormone-dependent cancer rates seen in Asian production in hLGC cultures are underway.
populations. However, to date we have studied only the F. jn.nutrition.org Another potential mechanism of endocrine modulation is vesiculosus species that is found in North America. Whereas the competitive inhibition of hormone receptors (39). Kelp brown seaweed is a major constituent of the Asian diet, the extract served as a competitive inhibitor to the binding of primary brown seaweeds consumed among Japanese popula- estradiol to both ER␣ and ER␤, with a slightly greater selec- tions include wakame (Unaria pinnatifida) and kombu (Lami- tivity toward ER␤ than ER␣. These findings suggest that naria japonica) and not F. vesiculosus. Further studies of the compounds found in F. vesiculosus could act as estradiol an- potential endocrine modulating effects of these more com- tagonists by decreasing the affinity of either ER␣ or ER␤ for its monly consumed seaweeds are needed along with dietary stud- ligand. Both receptors, which act as ligand-activated transcrip- ies of seaweed intake and cancer risk using nutritional epide- tion factors in target genes, are found in a wide variety of miology before firm conclusions can be made.
tissues. Despite the relatively similar binding affinities of ER␣ In summary, the detection of dietary components that have and ER␤ for estradiol, differences in binding specificity be- estrogen-reducing effects holds promise as a simple means of tween the ␣ and ␤ ERs and other ligands have been observed dietary modification to reduce risk of estrogen-dependent can- (40), although their disparate function in target tissues re- cers in the general population. Furthermore, the identification of the anti-estrogenic components in F. vesiculosus may lead to In the present study, there was no evidence of progestagenic the discovery of novel selective estrogen receptor modulators effects of dietary kelp administration in Sprague-Dawley rats, that may be useful in the treatment and/or prevention of and only small increases in progesterone were detected in estrogen-dependent cancers. To this end, the isolation and hLGC cultures following treatment with the kelp extract. Yet, identification of active components are currently in progress.
it was previously shown that F. vesiculosus administrationcorrected a progesterone deficiency in a woman with a lutealphase defect exhibiting high circulating estradiol levels (10).
These discrepancies may be due to the abnormally high estra- diol-to-progesterone ratio in the subject previously studied.
Percentage inhibition, IC50 and Ki values of kelp extracts There is evidence that estradiol, at relatively high but physi- (0.5, 5, and 50 ␮mol/L) on the binding of radioligands ologic concentrations, is a direct inhibitor of 3␤-hydroxy- steroid dehydrogenase, resulting in progesterone suppression in to their respective receptors (ER␣, ER␤, and PR-B) human luteal cells (41,42). Thus, in those with high circulat- in competitive radioligand binding assays ing estradiol, the anti-estrogenic activity of F. vesiculosus maynot only abrogate estradiol production, but also enhance pro- gesterone formation by alleviating estradiol’s inhibitory effects on 3␤-hydroxysteroid dehydrogenase. However, the potential impact of dietary kelp on circulating progesterone levels needsto be studied in a larger population.
One limitation of this study is that all rat blood was drawn during the morning of proestrus when estradiol levels were highest, but progesterone levels were not at their peak. Due to multiple blood draws from each rat, we were unable to obtain blood during other phases of the rat estrous cycle. However, we were able to determine the relative binding affinity of the kelp extract for PR-B, suggesting that kelp may act as a PR agonist.
Chemical analysis of F. vesiculosus has revealed that it contains many potentially bioactive compounds (43). Cur-rently, we are in the process of identifying which agents in F. vesiculosus are responsible for its anti-estrogenic activity. Pos- sible candidates include the bioactive polyphenols, sulfated 3 Negative values correspond to stimulation of binding.
ACKNOWLEDGMENTS
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We thank Satyabrata Nandi and Raphael Guzman for providing Breast cancer and hormone-replacement therapy in training and technical expertise in estrous cycling cytology determi- the Million Women Study. Lancet 362: 419 – 427.
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