Phytoestrogens include a wide variety of plant products that exert estrogenic and/or anti-estrogenic effects either inherently or after conversion by intestinal flora (Dwyer et al. 1994, Tang and Adams 1980). Initially, the importance of phytoestrogens was recognized by their effects on sheep fertility; more recently, they have come into prominence as a result of their possible anti-cancer properties in humans (Aldercreutz et al. 1993). Phytoestrogens also exhibit antioxidant, radical scavenging, hypolipidemic and serum cholesterol lowering properties (Franke et al. 1994).

There are many categories of phytoestrogens including lignans, isoflavonoids, coumestans and resorcyclic acid lactones, all of which bind estrogen receptors. However, phytoestrogens have a lower binding affinity than steroidal estrogens. Coumestans have ~0.05 and isoflavones 0.005 the binding affinity of estradiol for the estrogen receptor (Adams 1989). In addition to the lower binding affinity, the phytoestrogen-receptor complex formed is less stable and also less able to bind the DNA; this results in a shorter duration of binding to the DNA. These properties of stability and ability to be transformed also vary among the types of phytoestrogens (Adams 1989). Miksikek (1994) determined the binding affinity to the estrogen receptor of eight phytoestrogens; the order was as follows: zearalenone,-zearalanol, coumestrol, genistein, daidzein, phloetin, formonentin and biochanin A.

Thus, phytoestrogens are potentially important in the prevention of postmenopausal osteoporosis caused by estrogen deficiency. We have studied the effects of various phytoestrogens on bone and calcium metabolism using a well-described model of postmenopausal bone loss, the 6-mo-old oophorectomized rat (Dick et al. 1996, Hagaman et al. 1991, Wronski and Yen 1991).

This study examined the effects of two phytoestrogens, coumestrol and zearalanol, and a mixture of isoflavonoid phytoestrogens extracted from clover, on bone density and calcium metabolism. Coumestrol is a coumestan that has dietary importance because it is found in soybeans (Adams 1989, Lookhart 1979, Lundh et al. 1988, Wang et al. 1990), alfalfa sprouts (Franke et al. 1995, Livingstone et al. 1961, Lookhart 1979), large lima beans, red bean seeds, round split peas, mung bean sprouts and clover sprouts (Franke et al. 1995). Zearalanol, a resorcyclic acid lactone, was used previously to promote growth in beef cattle and thus used in the food industry. The clover contained a mixture of the isoflavones biochanin A, formonentin and genistein. These isoflavones are also present in many legumes.

MATERIALS AND METHODS

Table 1. Composition of diet for all studies1,2 [View Table]

All of the rats were oophorectomized at the start of the study. The surgery was conducted under pentobarbitone sodium (9 mg, Nembutal, Boehringer Ingelheim, NSW, Australia) and methoxyflurane, USP (Penthrane, Abbott Hospital Products, NSW, Australia) as necessary. The rats were then randomly allocated to the control group (n = 15), coumestrol-treated group (n = 15) or estrogen-treated group (n = 10). Postsurgery, the rats were treated for a period of 6 wk by intramuscular injection of cottonseed oil vehicle, cottonseed oil containing 1.5 µmol of coumestrol or 28.1 nmol estradiol, twice per week. Food was withheld from the rats while they were housed in metabolic cages for 48 h before oophorectomy and at 1 and 6 wk post-oophorectomy. Urine samples (24-h) were collected at 1 wk, and blood samples taken at 6 wk by cardiac puncture after food deprivation.

RESULTS

Table 2. Bone density measurements of femur, spine and whole body in rats before oophorectomy in three separate studies looking at the effect of the phytoestrogens, coumestrol and zearalanol, and a mixture of isoflavones on bone1 [View Table]

The calcium excreted in the urine was not correlated with the change in bone mineral density at any site. The change in calcium of the BMC was 1.8 ± 1.2 µmol/d, although the calcium excreted in the urine was only 0.33 ± 0.41 µmol/d. The calcium/creatinine ratio in urine was lower in the coumestrol- and estrogen-treated rats than in the oophorectomized controls at 1 wk (Table 3). The deoxypyridinoline/creatinine ratio and the pyridinoline/creatinine ratio were significantly higher in the oophorectomized control group than in the coumestrol- or estrogen-treated groups at 1 wk (P < 0.05). The deoxypyridinoline/creatinine ratio did not change significantly in the coumestrol- and estrogen-treated group (Table 3). At 6 wk, plasma cholesterol was significantly higher in the control group than in the coumestrol- and estrogen-treated groups (Table 3).

Table 3. Analysis of urine and plasma of rats in three separate studies looking at the effect of the phytoestrogens, coumestrol and zearalanol, and a mixture of isoflavones on bone before and 6 wk post-oophorectomy. Rats were untreated (controls) or were injected after oophorectomy with coumestrol (1.5 µmol) or estrogen (28.1 nmol) twice per week1,2 [View Table]

Table 4. Change in bone density 6 wk post-oophorectomy in untreated (controls), isoflavone fed or estrogen treated rats [View Table]

DISCUSSION

Although the mechanisms by which coumestrol and zearalanol reduced bone loss cannot be fully elucidated from these studies, the lower urinary calcium/creatinine ratio in the coumestrol group compared with the controls suggests that coumestrol treatment resulted in a decrease of calcium excretion. This decrease mirrored that observed with estrogen treatment and is consistent with the maintenance of bone mass in both groups. These data suggest a similar mechanism of effect of coumestrol and estrogen on the calcium balance in oophorectomized rats.

Because bone density was not correlated with the excretion of calcium in the urine and the calcium in the urine accounted for only a small amount of the calcium loss from the bones, this study suggests that coumestrol may also be affecting the calcium balance through mechanisms in the bowel. This would agree with studies of the effect of estrogen on calcium balance and would suggest that phytoestrogens affect calcium balance in the same way. It has previously been determined that the effects of estrogen deficiency on calcium balance in humans occur via a combination of decreased intestinal absorption and increased renal excretion (Heaney et al. 1978); recently it has been suggested that the major component of calcium balance to be affected in estrogen-deficienct growing rats is intestinal calcium secretion (Morris et al. 1995). This agrees with previous observations that at low calcium intakes, fecal calcium may exceed dietary calcium in humans (Kalser 1985), with the bowel as the primary route of calcium excretion in this instance.

The effect of coumestrol on bone resorption was determined by the measurement of pyridinoline and deoxypyridinoline. Pyridinoline and deoxypyridinoline are nonreducible cross-links derived from lysine and hydroxylysine residues and form only during the formation of mature collagen (Kent 1995). They are released from bone during bone resorption. Because estrogen and coumestrol inhibited the increase of pyridinoline and deoxypyridinoline in the urine that occurred in the oophorectomized controls, coumestrol inhibited at the resorption of bone. Thus, the reduction of bone loss is due at least in part to a decrease in bone resorption.

The lack of effect of the oral isoflavones probably indicates a lack of effective absorption because there was no effect on uterine weight or bone loss. Previous studies have shown that the isoflavones biochanin A and formonentin are very weak, exerting <0.006 and 0.0006, respectively, the potency of estradiol on alkaline phosphatase activity in a human endometrial cell line, compared with potencies of 0.084 for genistein, 0.061 for daidzein and 0.202 for coumestrol (Markiewicz et al. 1993). Measurement of affinity to the sheep uterine receptor showed agreement in the ranking of the compounds tested (Adams 1989). Other studies have previously demonstrated an effect of isoflavones on bone (Arjmandi et al. 1996). However, these previous studies were not performed on skeletally mature rats.

These data raise the question whether dietary phytoestrogens have potential for preventing bone loss in rats and humans. Because no dose-response studies have been performed on either coumestrol or zearalanol, it is not known if the amounts used in this study are required to achieve the results obtained. However, assuming an absorption efficiency of coumestrol of 0.2 as suggested by Adams (1989), these data indicate that the rat would have to consume 4 mg of coumestrol per week, an amount contained in 5.6 g of dried alfalfa sprouts or 0.7 g of dried clover sprouts (Franke et al. 1994). A rat would normally consume ~105 g of feed per week. A human would have to consume ~20 times the amount consumed by a rat, or 80 mg of coumestrol per week, an amount contained in 285.1 g of clover sprouts. However, the absorption studies performed have used the uterus and not bone as the measure of estrogenicity. Because the dose that will elicit the maximum effect is not yet known, more studies must be performed before it can be determined if this amount of sprouts is required.

Because postmenopausal osteoporosis is a condition characterized by the loss of bone density, the phytoestrogens coumestrol and -zearalanol may have a potential role in human health for preventing postmenopausal bone loss, while improving the plasma cholesterol concentration. Further studies must be done to determine whether coumestrol and -zearalanol are effective orally and what concentrations are required.