![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
|
|
|
|
|
||
,**
* Department of Pharmacology and Toxicology,
Department of Pathology and the
** Comprehensive Cancer Center, University of Alabama at Birmingham, Birmingham, AL 35294
3To whom correspondence should be addressed. E-mail: coral.lamartiniere{at}ccc.uab.edu.
 |
ABSTRACT |
|---|
 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
|---|
KEY WORDS: • aromatase • diethylstilbestrol • ERK • genistein • testis
Because of the proposed protective effects of phytoestrogens against osteoporosis, cardiovascular disease and hormone-dependent cancers in men and women, these compounds have been the targets of numerous investigations in recent years. Phytoestrogens are biologically active components present in numerous food products, and are normal constituents of a soy-based diet. Genistein, the predominant phytoestrogen in soy foods (1,2) and in the blood of individuals consuming a diet containing soy (3,4), is structurally similar to the endogenous estrogen, estradiol-17ß. Subsequent investigations have reported estrogen-like activity in receptor-binding assays and reporter gene studies in vitro, and in uterotrophic assays in vivo (5). Structural similarity and action through the estrogen receptors alpha and beta indicate the potential for estrogenic toxicity after exposure to high concentrations of genistein. In males in whom circulating estradiol concentrations are markedly lower than those in females, exposure to exogenous estrogens may be more likely to result in toxicity. Toxic effects of estrogenic compounds in males include reduction in circulating testosterone concentrations, impaired testicular descent, reproductive tract development and alterations to spermatogenesis and fertility (6). Despite the reported protective effects of genistein against disease, little is known regarding the potential for toxicity in developing males, particularly when administered at concentrations that are attainable in individuals on a soy-containing diet.
Exposure to estrogenic compounds during development interferes with growth and development of the reproductive tract in male rats. Previous studies showed that treatment with the synthetic estrogen diethylstilbestrol (DES) reduces androgen secretion and prostate size (7). In the testes, alterations to seminiferous tubule morphology (8–10) and disruption of spermatogenesis are associated with elevated apoptosis and reduced fertility in adulthood (9). An increase in apoptosis is also associated with tubule regression after androgen withdrawal in older rats (11). However, in an androgen-deprived state, there may also be direct effects of exogenous estrogens on germ cells expressing estrogen receptors (12–14). Atanassova et al. (9) reported that low doses of DES and other estrogenic compounds increase spermatocyte nuclear volume despite diminution of testicular development. Therefore, alterations to specific cell types may occur at low estrogen concentrations that fail to perturb androgen concentrations in the circulation, or after exposure to compounds with weak estrogenic activity, including genistein.
Recently we reported that exposure to genistein in the diet, starting at conception, does not alter early markers of reproductive maturation, including testicular descent (15). However, injections of pharmacological concentrations of genistein significantly reduces circulating testosterone concentrations (16,17), and alters testicular morphology and spermatogenesis (9). In contrast, exposure to dietary genistein from in utero development until young adulthood increases circulating testosterone concentrations (15), correlating to the increased circulating and tissue concentrations of genistein (15,17–20). The route of administration determines metabolism and disposition. Elevated testosterone concentrations may be indicative of altered steroidogenesis, suggesting the potential for subtle alterations to testicular development, morphology and spermatogenesis after dietary exposure.
Altered steroidogenesis in the testes of males exposed to genistein in the diet has yet to be determined. It is possible that changes in enzymes involved in converting testosterone to other steroidal metabolites may explain the observed elevation in circulating testosterone. Previous investigations showed that genistein binds to the active site of aromatase (21), the enzyme responsible for the final step of estrogen biosynthesis from C19 steroids, which suggests that this enzyme may be a target of genistein action in rats. Although in vitro studies indicated that phytoestrogens inhibit human placental aromatase activity in vitro (22), brain aromatase activity is not inhibited in rats fed a diet rich in phytoestrogens (23,24). The lack of altered aromatase activity in the brain, which might explain altered testosterone concentrations resulting from a brain-specific mechanism, does not exclude a similar mechanism that may occur in tissues. However, in vivo effects of genistein on aromatase activity in male reproductive tissues, particularly the testes, remain to be determined.
In this study, we investigated the potential for estrogen-related toxicity in the testes of male rats fed genistein in the diet at nutritionally relevant concentrations. We compared effects to the known developmental and male reproductive toxicant, DES, with particular emphasis on testicular weight, morphology, androgen receptor (AR), epidermal growth factor receptor (EGFR) and extracellular signal-related kinase (ERK) expression after dietary exposure during early development. We also investigated the effects on testicular aromatase activity to identify alterations to early male reproductive development.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
|---|
Animal care and treatment were performed according to established guidelines approved by the UAB Animal Care Committee. Sprague-Dawley CD rats were obtained from Charles River Breeding Laboratories (Raleigh, NC) and housed in a climate-controlled room with a 12-h light/12-h dark cycle. Rats were bred and fed a phytoestrogen-free AIN-76A diet (25) (Harlan Teklad, Madison, WI) at birth. Litters were adjusted so that there were 10 offspring per dam. Offspring were weaned on d 21 postpartum and males were separated into treatment groups.
For dietary treatment, chemically synthesized genistein (98.5% pure; Hoffmann-LaRoche, Basel, Switzerland) was added to the AIN-76A diet for concentrations of 0, 250 or 1000 mg/kg diet. Rats fed the 250 mg genistein/kg diet had serum concentrations that approximate physiological levels in humans, whereas 1000 mg genistein/kg diet resulted in concentrations that are at the upper limit found in humans consuming soy products (3,15,26). Rats fed either of the genistein-containing diets did not have altered food intake or body weights compared with rats not exposed to genistein (15,27). Rats were fed genistein or DES (75 µg/kg diet; Sigma, St. Louis, MO) from d 21 to d 35 postpartum and killed by decapitation on d 35. DES was selected for known effects on the male reproductive tract (28). At the time of killing, the testes were removed and weights recorded. A section from the middle of one testis was fixed in formalin for morphological and immunohistochemical analysis, whereas an additional piece from the same testis was frozen for subsequent Western blot analysis. A portion of the other testis was immediately processed for measurement of aromatase activity.
Testis morphology.
After overnight fixation in formalin, the testes were dehydrated in an alcohol gradient and placed in xylene. Tissues were then blocked in paraffin, cut into 5-µm sections and stained with hematoxylin and eosin. Tubule dimensions were investigated according to a previous report of reduced testicular tubule dimensions after DES treatment (9). Testicular sections were photographed using a Nikon Coolpix 990 digital camera (Nikon, Melville, NY) mounted on the microscope. Tubular dimensions were measured for four tubules per section for n = 1. Four separate sections from each testis were analyzed for a total of 16 tubules per testis. Measurements were completed on four rats per treatment group.
Western blot analysis.
Western blot analysis for EGFR, ERK and phosphorylated ERK (P-ERK) was carried out as previously described (29). The same procedure was used for AR (N-20; Santa Cruz Biotechnology, Santa Cruz, CA). Appropriate control peptides and molecular weight markers were used to identify the proteins of interest.
Immunohistochemical analysis.
Sections (5 µm) were taken from the paraffin blocks described above, deparaffinized and rehydrated. Endogenous peroxidase activity was blocked by use of hydrogen peroxide, and AR and P-ERK epitope exposures were enhanced by boiling in antigen unmasking solution (Vector Laboratories, Burlingame, CA). Slides were incubated overnight at 4°C in a humidity chamber with the appropriate antibody. AR and P-ERK antibodies were the same used for Western blot analysis (described above), and the EGFR antibody (Santa Cruz Biotechnology) cross-reacted with the rat epitope. Detection was performed by use of a biotinylated secondary antibody conjugated to streptavidin peroxidase, followed by chromogen detection with NOVA red substrate (Vector Laboratories) with hematoxylin counterstain.
Proliferating cell nuclear antigen (PCNA; Zymed, South San Francisco, CA) was analyzed according to the manufacturer’s protocol. Variations to methodology described for AR, P-ERK and EGFR immunostaining include only 1 h incubation with primary antibody and use of 3,3'diaminobenzidene as a chromogen. Sections were analyzed for cellular distribution and relative staining intensity as described previously (30). For detection of apoptosis, DNA fragment end labeling was performed by use of terminal deoxynucleotidyl transferase, which recognizes fragmented or apoptotic nuclei according to the manufacturer’s protocol (Oncogene Research Products, Boston, MA). Three separate slides were analyzed for each testis and the average number of seminiferous tubules with extensive apoptosis (arbitrarily established as >10% of total nuclei) was calculated. For all markers, normal serum was used instead of primary antibody as negative controls and positive control tissues allowed for only staining above background to be considered for analysis.
Aromatase activity.
Testicular aromatase activity was determined by measurement of tritiated water released after conversion of testosterone labeled in the 1 beta position to estradiol, based on the methodology described by Lephart and Ojeda (31). Reaction conditions and incubation times were optimized for the testes from 35-d-old rats. Reactions were incubated for 2 h at 37°C while rotating, then terminated and processed as described elsewhere (31). The enzymatic rate was adjusted to account for changes in the aqueous phase volume and calculated as pmol/(min · mg protein), and expressed as the percentage of the mean control activity. Assay conditions yielded zero-order kinetics. Control reactions included adult testicular homogenates and addition of unlabeled testosterone to test samples, and a blank reaction (without source of aromatase) to control for background radioactivity.
Aromatase mRNA expression.
RT-PCR analysis was performed as previously described (32) by use of the following primers for aromatase: primer 1, 5'-TGCTGGACACTTCTAACACG-3'; primer 2, 5'-GATACTCTGCGATGAGAAGC-3', forming a 395-bp fragment.
Testicular sex steroid concentrations.
Testicular steroid concentrations were determined according to methods described by Nnane et al. (33) and Oi et al. (34), with modifications. Samples were homogenized in a 5X volume of 20 mmol/L potassium phosphate buffer (pH 7.4). After homogenization, samples were centrifuged briefly at 800 x g to remove cell debris. The supernatant was removed and an equal volume of buffer was added to the pellet. Three series of ethyl acetate extractions were used to isolate the remaining steroids from the pellet. The pooled ethyl acetate was dried under a gentle stream of air and the precipitate combined with the supernatant. Preliminary analysis using tritiated testosterone demonstrated that recovery was >99% by use of this approach. Testosterone and estradiol concentrations were measured by use of an in-house radioimmunoassay according to the manufacturer’s protocols (DSL, Webster, TX).
Statistical analysis.
Mean values for body and reproductive tract weights, serum androgen concentrations and protein band intensity were compared through the use of one-way ANOVA followed by the Tukey test (Sigma Stat; Jandel Scientific, San Rafael, CA). Differences were considered significant at P < 0.05.
|
RESULTS |
|---|
|
TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
|---|
Testicular weights were significantly reduced in rats fed the synthetic estrogen DES from d 21 to d 35, whereas testes weights were not affected by consumption of genistein-containing diets (Table 1). A reduction in testicular weights was associated with a proportional reduction in the dimensions of the seminiferous tubules. Genistein did not significantly alter tubule dimensions, consistent with the lack of effect on weights.
|
Western blot analysis demonstrated that AR protein expression was reduced in a dose-dependent manner in rats fed genistein in the diet, although expression was not significantly lower than that in controls (Table 2). On the other hand, exposure to DES in the diet resulted in 50% less AR protein in the testes after the 2-wk exposure (P < 0.05). AR immunolocalization in all treatment groups revealed staining, predominantly in Sertoli and interstitial cells, with markedly lighter staining intensity in spermatocytes and spermatids, whereas spermatogonia had no immunostaining (data not shown). In the testes of rats fed DES, the reduction in AR protein concentrations determined by Western blot analysis corresponded with drastic reduction in Sertoli cell immunostaining, with considerable but less dramatic reduction in intensity in Leydig cells. AR staining in rats fed genistein in the diet did not differ in intensity from that of control testes. In DES-treated rats, there were also fewer spermatids per tubule and subsequently less apparent immunostaining. This was in contrast to rats fed genistein, for which there was no alteration to sperm development.
|
Testes of DES-treated rats had significantly higher concentrations of EGFR and its downstream targets, ERK 1 and ERK 2 (Table 2). The amounts of P-ERK 1 and P-ERK 2 were also increased by DES treatment. There were no significant effects of genistein on EGFR, ERK expression or ERK phosphorylation. Immunohistochemical localization of the EGFR revealed staining, predominantly in spermatocytes in rats from all treatment groups (data not shown). Although there were no differences in EGFR expression in the testes of rats fed genistein in the diet, relative staining intensity compared to that of controls was greater in rats fed DES in the diet.
Apoptosis, cell proliferation and P-ERK expression.
Because spermatocyte accumulation and reduction in testicular size after DES exposure was observed, we analyzed the prevalence of apoptotic nuclei after exposure to DES or genistein in the diet. About 70% of all seminiferous tubules in rats fed DES had elevated apoptosis (>10% of total nuclei counted) (Table 1). This was in contrast to only 10% of control tubules undergoing a similar degree of apoptosis. A majority of the apoptotic nuclei in rats fed DES in the diet were spermatocytes or spermatogonia. On the other hand, the incidence of apoptosis in tubules of rats fed genistein in the diet was similar to that found in rats fed the control diet.
To determine whether changes in growth factor signaling correlated with proliferation or degeneration of developing sperm, serial sections were stained for PCNA, P-ERK and apoptotic cells (Fig. 1). PCNA staining was located in germ cells of testes from control and genistein-fed rats. In rats fed DES in the diet, more layers of PCNA-labeled spermatocytes were detected than in controls.
|
Sex steroid metabolism.
Testosterone concentrations tended to be greater (P < 0.546) and estradiol concentrations tended to be lower (P < 0.793) in testicular homogenates of rats fed genistein in the diet compared with controls (Table 3). The ratio of estradiol to testosterone was reduced over 66% in rats fed the 1000 mg genistein/kg diet compared to controls. Therefore, we analyzed the effects of genistein on aromatase activity as a possible explanation for this effect. Aromatase activity in testicular homogenates was reduced in a dose-dependent manner by genistein, with a 20 and 25% reduction in activity in rats fed the 250 and 1000 mg genistein/kg diet, respectively (P < 0.05) (Table 4). A comparable reduction in mRNA expression for aromatase was also demonstrated by RT-PCR. Because the testes weight was drastically reduced and circulating androgens were minimal compared with controls, aromatase activity and mRNA expression were not investigated in rats fed DES in the diet.
|
|
|
DISCUSSION |
|---|
|
TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
|---|
Exposure to estrogenic compounds during early periods of development has been associated with adverse effects on male reproductive health (6). In this report we investigated the potential for testicular toxicity in male rats exposed to genistein, compared with DES, in the diet for 2 wk during early pubertal development. No significant effects were observed on testes weight and morphology, and the rate of apoptosis and cell proliferation in rats fed 250 and 1000 mg genistein/kg diet compared to controls. A similar lack of toxicity was previously reported after exposure to lower concentrations of genistein in the diet (5 mg/kg) (35). However, these observations are in contrast to a study implicating genistein as a testicular toxicant when administered by injection of 4 mg/(kg · d) (9). Atanassova et al. (9) reported increased apoptosis accompanied by an increase in spermatocyte/Sertoli nuclear volume, and a reduction in lumen volume, similar to the effects seen in DES-treated rats. In our study, we did not observe spermatocyte accumulation or increased apoptosis after exposure to genistein in the diet. The lack of testicular toxicity from lifetime exposure (in utero until d 70) to genistein in the diet is further supported by our observation of sperm counts and motility that are similar for rats fed 25 and 250 mg genistein/kg diet and control rats (W. A. Fritz and C. A. Lamartiniere, unpublished results). Also, there was a lack of effect of genistein on AR, EGFR and ERK protein concentrations in testes when administered in the diet. Similar effects of slight, but not significant, decreases in AR expression in the prostate of rats were previously reported (15).
The most probable explanation for the difference in genistein effects on testes toxicity in rats is absorption and metabolism. Andlauer and colleagues (36,37) demonstrated that a majority of the genistein absorbed across the intestines is conjugated before entering the circulation, and further conjugation occurs through enterohepatic circulation (38). In experiments by Atanassova et al. (9), in which they showed changes in testes weights and alterations to the lumen and spermatogenesis, genistein was administered by injection. In our studies we administered genistein in the diet, the natural route of human and animal consumption. We previously demonstrated that injection of genistein results in 
46% of circulating total genistein being free genistein 24 h after injection of rats (18). Although Atanassova et al. (9) did not report circulating genistein concentrations, we determined that rats fed 250 mg genistein/kg diet have 1.785 nmol genistein/L serum, whereas consumption of the 1000 mg/kg diet resulted in total circulating concentrations of 9.640 nmol/L serum (15). However, >98% of the total genistein identified in the circulation after dietary exposure was conjugated (15), rather than in the free, biologically active form that enters the circulation after injection. The bioavailability, or concentration of unconjugated genistein responsible for biological response, of injected genistein is substantially greater than that of oral genistein (23-fold). The presence of predominantly glucuronidated genistein is a probable explanation for the lack of testicular toxicity after ingestion of genistein in the diet, compared with exposure by injection, where all of the genistein is initially unconjugated.
Interestingly, genistein in the diet did result in slightly increased testosterone concentrations and slightly decreased estradiol concentrations in the testes, and these changes were dose dependent. This resulted in decreased estrogen-to-testosterone concentration ratios, including a 66% decrease at the highest dietary genistein dose (1000 mg genistein/kg diet). We previously reported slightly increased testosterone, but not dihydrotestosterone, concentrations in the blood of adult rats and mice treated with genistein in the diet (15,19,39). To explain this alteration in steroidogenesis, we measured aromatase, the enzyme that converts androgen to estrogen. We found that aromatase activity was inhibited in the testes of rats fed the diet containing the highest concentrations of genistein (1000 mg/kg diet). Consistent with this finding, we observed that dietary exposure to genistein also downregulates aromatase mRNA in the testes, although it remains to be elucidated whether inhibition of aromatase activity is a result of competition for the active site by increasing concentrations of genistein that occur with greater dietary concentrations (19), or whether reducing enzyme expression regulates activity. It is also uncertain whether the increase in testosterone concentrations observed after ingestion of genistein is a direct result of aromatase inhibition, as previously demonstrated by use of aromatase inhibitors (40), or whether aromatase inhibition by genistein occurs in response to elevated testosterone concentrations.
Based on the knowledge that prostate cancer develops in men as they age and when androgen concentrations are declining, it has been suggested that maintenance of normal concentrations of testosterone may actually prevent development of prostate cancer by minimizing androgen-independent growth, for which there is little successful therapy (41). Maintenance of slightly elevated androgens in the presence of estrogenic compounds, including genistein, could explain the lack of estrogenic toxicity associated with feedback reduction in testosterone, and allows prostate growth to be regulated by androgens. Thus, the health benefits of genistein in the prevention of prostate cancer in men consuming soy (42) could be partly a result of androgen regulation at the level of the testes, maintaining sufficient androgen concentrations to regulate growth and function at a period when disease otherwise becomes predominant.
DES.
In contrast to the lack of effect of genistein for toxicity in rats, those fed DES in the diet had reduced testicular weights and impaired seminiferous tubule morphology, confirming previous findings that reduction in testicular weight by DES is associated with adverse effects on seminiferous tubule development (8–10). In our investigation DES exposure was also associated with accumulation of spermatocytes in most, if not all, of the individual tubules, indicative of impaired sperm maturation. It is likely that impaired spermatogenesis after DES exposure was at least partially a result of reduced testosterone concentration, as demonstrated in previous studies (15,28). Testosterone stimulates Sertoli cells to regulate sperm maturation, and interference with this action is evident by reduction in Sertoli cell AR immunostaining after DES exposure. However, it is also possible that spermatocyte accumulation in DES-treated rats is attributed to increased proliferation. In rats the first wave of spermatogenesis follows a specific developmental progression. Growth and expansion of the seminiferous tubules, and an increase in the number and volume of germ cells is followed by a reduction in the number of degenerating sperm as maturation occurs (43,44). We observed a combination of elevated PCNA in spermatocytes and apoptosis in late spermatocytes and spermatids of DES-treated rats, reflective of rapid cell turnover in the testes (45). The result is arrested sperm development, perhaps because of the lack of testosterone stimulation. EGFR expression was increased in the testes after exposure to DES. Initially, it was unclear whether this increase was merely a result of accumulated spermatocytes that express this receptor, or whether the elevation was associated with extensive apoptosis, as previously suggested (46). It is apparent from those studies and ours that accumulation of spermatocytes occurred in spite of an increased apoptotic index and tubule regression (11).
Through the use of serial sections, we found that tubules in all treatment groups with the greatest extent of apoptosis did not have the most intense P-ERK staining; rather, tubules with elevated P-ERK showed the most PCNA staining. The association between PCNA and P-ERK staining suggests that ERK function as regulators of proliferation during sperm maturation in developing testes. Previous studies demonstrated that nuclear ERK1/2 in spermatogonia and spermatocytes is associated with proliferation in frogs during seasonal increases in spermatogenesis (47). The predominantly nuclear P-ERK staining in Sertoli cells of control and treated rats suggests active signaling, given that ERK have many nuclear substrates (48). In contrast, other cell types including spermatogonia and spermatocytes showed predominantly perinuclear staining. MAP-2, a possible substrate for ERK, was localized to the germ cell cytoplasm (49), suggesting that activated ERK may phosphorylate cytoplasmic targets in the testes.
Similar to the observed morphological and molecular effects and increase in EGFR expression after DES treatment in the current investigation, EGF overexpression in mice is associated with active meiosis and impaired spermatogenesis, reducing formation of spermatids and spermatozoa (50). Spermatogonia isolated from frogs during a minimally proliferative period can be stimulated to proliferate by estradiol, an effect blocked by the antiestrogen, ICI 182,780 (47). This suggests that estrogenic compounds can regulate spermatogonial proliferation either by direct effects on estrogen receptors (12–14) that stimulate germ cell proliferation, or by interaction between the estrogen receptor and colocalized EGFR (51) in the spermatogonia through a cross-talk mechanism (52).
In summary, we have demonstrated that exposure to genistein in the diet did not adversely affect the structure or function of the testes, even when administered to rats at concentrations that result in circulating concentrations similar to those found in infants consuming soy formula (26). Genistein concentrations in the diet were 3333 to 13,333 higher compared to those of DES, illustrating the weak effector properties of genistein for toxicity. On the other hand, high concentrations of genistein in the diet inhibited conversion of testosterone to estradiol in testicular homogenates, consistent with reduction of the estrogen-to-testosterone ratio in the testes of rats fed genistein. Modulation of testicular aromatase may be an important mechanism to account for the health benefits of a soy-containing diet, and suggests a potential mode of action that contributes to the lower incidence of clinical prostate cancer in men consuming soy. This is of considerable importance in that unlike the rat, the human prostate contains aromatase, and that estrogens, in combination with androgens, are involved in development of prostate cancer (53,54).
|
FOOTNOTES |
|---|
2 Portions of this research were presented at the annual meeting of the American Association of Cancer Research [Fritz, W. A., Wang, J. & Lamartiniere, C. A. (2001) Dietary genistein reduces expression of the steroid biosynthetic enzymes, aromatase and 5alpha-reductase in the male rat. Proc. Amer. Assoc. for Cancer Res. 42: 461, 2001.].
4 Abbreviations used: AR, androgen receptor; DES, diethylstilbestrol; EGFR, epidermal growth factor receptor; ERK, extracellular signal-regulated kinase; PCNA, proliferating cell nuclear antigen; P-ERK, phosphorylated extracellular signal-regulated kinase.
Manuscript received 20 December 2002. Initial review completed 24 January 2003. Revision accepted 6 March 2003.
|
LITERATURE CITED |
|---|
|
TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
|---|
1. Murphy, P. A. (1982) Phytoestrogen content of processed soybean products. Food Technol. 34:60-64.
2. Coward, L., Narnes, N. C., Setchell, K.D.R. & Barnes, S. (1993) Genistein and polyphenols, and their beta-glycoside conjugates: anti-tumor isoflavones in soybean foods from American and Asian diets. J. Agric. Food Chem. 41:1961-1967.
3. Adlercreutz, H., Markkanen, H. & Watanabe, S. (1993) Plasma concentrations of phyto-estrogens in Japanese men. Lancet 342:1209-1210.[Medline]
4. Morton, M. S., Chan, P.S.F., Cheng, C., Blacklock, N., Matos-Ferreira, A., Abranches-Monteiro, L., Correia, R., Lloyd, S. & Griffiths, K. (1997) Lignans and isoflavonoids in plasma and prostatic fluid in men: samples from Portugal, Hong Kong, and the United Kingdom. Prostate 32:122-128.[Medline]
5. Boulker, K. B. & Hilakivi-Clarke, L. (2000) Genistein: does it prevent or promote breast cancer?. Environ. Health Perspect. 108:701-708.[Medline]
6. Safe, S. (2000) Endocrine disruptors and human health—is there a problem?. An update. Environ. Health Perspect. 108:487-493.[Medline]
7. Yamashita, A., Hayashi, N., Sugimura, Y., Cunha, G. R. & Kawamura, J. (1996) Influence of diethylstilbestrol, leuprolelin (a luteinizing hormone-releasing hormone analog), finasteride (a 5 alpha-reductase inhibitor), and castration on the lobar subdivisions of the rat prostate. Prostate 29:1-14.[Medline]
8. Sharpe, R. M., Atanassova, N., McKinnell, C., Parte, P., Turner, K. J., Fisher, J. S., Kerr, J. B., Groome, N. P., Macpherson, S., Millar, M. R. & Saunders, P. T. (1998) Abnormalities in functional development of the Sertoli cells in rats treated neonatally with diethylstilbestrol: a possible role for estrogens in Sertoli cell development. Biol. Reprod. 59:1084-1094.
9. Atanassova, N., McKinnell, C., Turner, K. J., Walker, M., Fisher, J. S., Morley, M., Millar, M. R., Groome, N. P. & Sharpe, R. M. (2000) Comparative effects of neonatal exposure of male rats to potent and weak (environmental) estrogens on spermatogenesis at puberty and the relationship of adult testis size and fertility: evidence for stimulatory effects of low estrogen levels. Endocrinology 141:3898-3907.
10. McKinnell, C., Atanassova, N., Williams, K., Fisher, J. S., Walker, M., Turner, K. J., Saunders, T. K. & Sharpe, R. M. (2001) Suppression of androgen action and the induction of gross abnormalities of the reproductive tract in male rats treated neonatally with diethylstilbestrol. J. Androl. 22:323-338.[Abstract]
11. Nonclercq, D., Reverse, D., Toubeau, G., Beckers, J. F., Sulon, J., Laurent, G., Zanen, J. & Heuson-Stiennon, J. A. (1996) In situ demonstration of germinal cell apoptosis during diethylstilbestrol-induced testis regression in adult male Syrian hamsters. Biol. Reprod. 55:1368-1376.[Abstract]
12. Saunders, P.T.K., Maguire, S. M., Gaughan, J. & Millar, M. R. (1997) Expression of estrogen receptor beta (ER beta) in multiple rat tissues. J. Endocrinol. 154:R13-R16.
13. Saunders, P.T.K., Fisher, J. S., Sharpe, R. M. & Millar, M. R. (1998) Expression of oestrogen receptor beta (ERb) occurs in multiple cell types, including some germ cells, in the rat testis. J. Endocrinol. 156:R13-R17.[Abstract]
14. van Pelt, A. M., de Rooij, D. G., van der Burg, B., van der Saag, P. T., Gustafsson, J. A. & Kuiper, G. G. (1999) Ontogeny of estrogen receptor-beta expression in rat testis. Endocrinology 140:478-483.
15. Fritz, W. A., Wang, J., Eltoum, I.-E. & Lamartiniere, C. A. (2002) Dietary genistein down-regulates androgen and estrogen receptor expression in the rat prostate. Mol. Cell. Endocrinol. 186:89-99.[Medline]
16. Strauss, L., Makela, S., Joshi, S., Huhtaniemi, I. & Santti, R. (1998) Genistein exerts estrogen-like effects in male mouse reproductive tract. Mol. Cell. Endocrinol. 144:83-93.[Medline]
17. Schleicher, R. L., Lamartiniere, C. A., Zheng, M. & Zhang, M. (1999) The inhibitory effect of genistein on the growth and metastasis of a transplantable rat accessory sex gland carcinoma. Cancer Lett. 136:195-201.[Medline]
18. Cotroneo, M. S. & Lamartiniere, C. A. (2001) Pharmacologic, but not dietary, genistein supports endometriosis in a rat model. Toxicol. Sci. 61:68-75.
19. Dalu, A., Haskell, J. F., Coward, L. & Lamartiniere, C. A. (1998) Genistein, a component of soy, inhibits the expression of the EGF and ErbB/Neu receptors in the rat dorsolateral prostate. Prostate 37:36-43.[Medline]
20. Fritz, W. A., Coward, L., Wang, J. & Lamartiniere, C. A. (1998) Dietary genistein: perinatal mammary cancer prevention, bioavailability and toxicity testing in the rat. Carcinogenesis 19:2151-2158.
21. Kao, Y.-C., Zhou, C., Sherman, M., Laughton, C. A. & Chen, S. (1998) Molecular basis of the inhibition of human aromatase (estrogen synthetase) by flavone and isoflavone phytoestrogens: a site-directed mutagenesis study. Environ. Health Perspect. 106:85-92.[Medline]
22. Pelissero, C., Lenczowski, M. J., Chinzi, D., Davail-Cuisset, B., Sumpter, J. P. & Fostier, A. (1996) Effects of flavonoids on aromatase activity, an in vitro study. J. Steroid Biochem. Mol. Biol. 57:215-223.[Medline]
23. Weber, K. S., Jacobson, N. A., Setchell, K. D. & Lephart, E. D. (1999) Brain aromatase and 5alpha-reductase, regulatory behaviors and testosterone levels in adult rats on phytoestrogen diets. Proc. Soc. Exp. Biol. Med. 221:131-135.[Medline]
24. Weber, K. S., Setchell, K.D.R. & Lephart, E. D. (2001) Maternal and perinatal brain aromatase: effects of dietary soy phytoestrogens. Dev. Brain Res. 126:217-221.[Medline]
25. Bieri, J. G., Stoewsand, G. S., Briggs, G. M., Phillips, R. W., Woodward, J. C. & Knapka, J. J. (1980) Report of the American Institute of Nutrition Ad Hoc Committee on Standards for Nutritional Studies. J. Nutr. 110:1340-1348.
26. Setchell, K. D., Zimmer-Nechemias, L., Cai, J. & Heubi, J. E. (1997) Exposure of infants to phyto-oestrogens from soy-based infant formula. Lancet 350:23-27.[Medline]
27. Flynn, K. M., Ferguson, S. A., Delclos, K. B. & Newbold, R. R. (2000) Effects of genistein exposure on sexually dimorphic behaviors in rats. Toxicol. Sci. 55:311-319.
28. Gibson, J. P., Newberne, J. W., Kuhn, W. L. & Elsea, J. R. (1967) Comparative chronic toxicity of three oral estrogens in rats. Toxicol. Appl. Pharmacol. 11:489-510.[Medline]
29. Cotroneo, M. S., Wang, J., Fritz, W. A., Eltoum, I.-E. & Lamartiniere, C. A. (2002) Genistein action in the prepuberatal mammary gland in a chemoprevention model. Carcinogenesis 23:1467-1474.
30. Brown, N. M., Wang, J., Cotroneo, M. S., Zhao, Y.-X. & Lamartiniere, C. A. (1998) Prepubertal genistein treatment modulates TGF-alpha, EGF, and EGF-receptor mRNAs and proteins in the rat mammary gland. Mol. Cell. Endocrinol. 144:149-165.[Medline]
31. Lephart, E. D. & Ojeda, S. R. (1990) Hypothalamic aromatase activity in male and female rats during juvenile peripubertal development. Neuroendocrinology 51:385-393.[Medline]
32. Fritz, W. A., Eltoum, I. E., Cotroneo, M. S. & Lamartiniere, C. A. (2002) Genistein alters growth but is not toxic to the rat prostate. J. Nutr. 132:3007-3011.
33. Nnane, I. P., Kato, K., Liu, Y., Long, B. J., Lu, Q., Wang, X., Ling, Y. Z. & Brodie, A. (1999) Inhibition of androgen synthesis in human testicular and prostatic microsomes and in male rats by novel steroidal compounds. Endocrinology 140:2891-2897.
34. Oi, Y., Imafuku, M., Shishido, C., Kominato, Y., Nishimura, S. & Iwai, K. (2001) Garlic supplementation increases testicular testosterone and decreases plasma corticosterone in rats fed a high protein diet. J. Nutr. 131:2150-2156.
35. Roberts, D., Veeramachaneni, D. N., Schlaff, W. D. & Awoniyi, C. A. (2000) Effects of chronic dietary exposure to genistein, a phytoestrogen, during various stages of development on reproductive hormones and spermatogenesis in rats. Endocrine 13:281-286.[Medline]
36. Andlauer, W., Kolb, J., Stehle, P. & Fürst, P. (2000) Absorption and metabolism of genistein in isolated rat small intestine. J. Nutr. 130:843-846.
37. Andlauer, W., Kolb, J. & Fürst, P. (2000) Absorption and metabolism of genistein in isolated rat small intestine. FEBS Lett. 475:127-130.[Medline]
38. Sfakianos, J., Coward, L., Kirk, M. & Barnes, S. (1997) Intestinal uptake and biliary excretion of the isoflavone genistein in rats. J. Nutr. 127:1260-1268.
39. Mentor-Marcel, R., Lamartiniere, C. A., Greenberg, N. & Elagavish, A. (2001) Genistein in the diet reduces the incidence of prostate tumors in a transgenic mouse (TRAMP). Cancer Res. 61:6777-6782.
40. Shetty, G., Krishnamurthy, H., Krishnamurthy, H. N., Bhatnagar, A. S. & Moudgal, N. R. (1998) Effect of long-term treatment with aromatase inhibitor on testicular function of adult male bonnet monkeys (M. radiata). Steroids 63:414-420.[Medline]
41. Prehn, R. T. (1999) On the prevention and therapy of prostate cancer by androgen administration. Cancer Res. 59:4161-4164.
42. Adlercreutz, H. (1995) Phytoestrogens: epidemiology and a possible role in cancer protection. Environ. Health Perspect. 103:103-112.
43. Russell, L. D., Alger, L. E. & Nequin, L. G. (1987) Hormonal control of pubertal spermatogenesis. Endocrinology 120:1615-1632.
44. Russell, L. D., Bartke, A. & Goh, J. C. (1989) Postnatal development of the Sertoli cell barrier, tubular lumen, and cytoskeleton of Sertoli and myoid cells in the rat, and their relationship to tubular fluid secretion and flow. Am. J. Anat. 184:179-189.[Medline]
45. Sarica, K., Bakir, K., Erbagci, A., Yagci, F., Uysal, O. & Ucak, R. (2001) Unilateral testicular torsion: evaluation of bcl-2, p-53 and PCNA expression in contralateral testes. Urol. Int. 66:94-99.[Medline]
46. Kyriakis, J. M. (1999) Making the connection: coupling of stress-activated ERK/MAPK (extracellular-signal-regulated kinase/mitogen-activated protein kinase) core signalling modules to extracellular stimuli and biological responses. Biochem. Soc. Symp. 64:29-48.[Medline]
47. Chieffi, P., Colucci-D’Amato, G. L., Staibano, S., Franco, R. & Tramontano, D. (2000) Estradiol-induced mitogen-activated protein kinase (extracellular signal-related kinase 1 and 2) activity in the frog (Rana esculenta) testis. J. Endocrinol. 167:77-84.[Abstract]
48. Seger, R. & Krebs, E. G. (1995) The MAPK signaling cascade. FASEB J. 9:726-735.[Abstract]
49. Loveland, K. L., Hayes, T. M., Meinhardt, A., Zlatic, K. S., Parvinen, M., de Kretser, D. M. & McFarlane, J. R. (1996) Microtubule-associated protein-2 in the rat testis: a novel site of expression. Biol. Reprod. 54:896-904.[Abstract]
50. Wong, R. W., Kwan, R. W., Mak, K. K., Sham, M. H. & Chan, S. Y. (2000) Overexpression of epidermal growth factor induced hypospermatogenesis in transgenic mice. J. Biol. Chem. 275:18297-18301.
51. Caussanel, V., Tabone, E., Mauduit, C., Dacheux, F. & Benahmed, M. (1996) Cellular distribution of EGF, TGFalpha and their receptor during postnatal development and spermatogenesis of the boar testis. Mol. Cell. Endocrinol. 123:61-69.[Medline]
52. Weigel, N. L. & Zhang, Y. (1998) Ligand-independent activation of steroid hormone receptors. J. Mol. Med. 76:469-479.[Medline]
53. Bosland, M. C., Ford, H. & Horton, L. (1995) Induction at high incidence of ductal prostate adenocarcinomas in NBL/Cr and Sprague-Dawley Hsd: SD rats treated with a combination of testosterone and estradiol 17 beta or diethylstilbestrol. Carcinogenesis 16:1311-1317.
54. Pollard, M., Snyder, D. L. & Luckert, P. H. (1987) Dihydrotestosterone does not induce prostate adenocarcinoma in L-W rats. Prostate 10:325-331.[Medline]
This article has been cited by other articles:
|
|
 |
|
  C. Montani, M. Penza, M. Jeremic, G. Biasiotto, G. La Sala, M. De Felici, P. Ciana, A. Maggi, and D. Di Lorenzo Genistein is an Efficient Estrogen in the Whole-Body throughout Mouse Development Toxicol. Sci., May 1, 2008; 103(1): 57 - 67. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. F. M. Mikkila, J. Toppari, and J. Paranko Effects of Neonatal Exposure to 4-Tert-Octylphenol, Diethylstilbestrol, and Flutamide on Steroidogenesis in Infantile Rat Testis Toxicol. Sci., June 1, 2006; 91(2): 456 - 466. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. A.L. Tan, M. Walker, K. Morris, I. Greig, J. I. Mason, and R. M. Sharpe Infant feeding with soy formula milk: effects on puberty progression, reproductive function and testicular cell numbers in marmoset monkeys in adulthood Hum. Reprod., April 1, 2006; 21(4): 896 - 904. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. E. Juan, E. Gonzalez-Pons, T. Munuera, J. Ballester, J. E. Rodriguez-Gil, and J. M. Planas trans-Resveratrol, a Natural Antioxidant from Grapes, Increases Sperm Output in Healthy Rats J. Nutr., April 1, 2005; 135(4): 757 - 760. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. E. Wood, T. C. Register, M. S. Anthony, N. D. Kock, and J. M. Cline Breast and Uterine Effects of Soy Isoflavones and Conjugated Equine Estrogens in Postmenopausal Female Monkeys J. Clin. Endocrinol. Metab., July 1, 2004; 89(7): 3462 - 3468. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
