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Alterations in Clinically Important Phytoestrogens in 
Genetically Modified, Herbicide-Tolerant Soybeans 

Journal of Medicinal Food v.1, n. 4, 1jul99

Marc A. Lappé, Ph.D., Center for Ethics and Toxics, Gualala CA E. Britt Bailey, M.A., Center for Ethics and Toxics, Gualala, CA Chandra Childress, M.S., Children's Hospital Medical Center, Cincinnati, OH Kenneth D.R. Setchell, Ph.D., Children's Hospital Medical Center, Cincinnati, OH

Contact: Dr. Marc Lappé PO Box 673, Gualala, CA 95445 mlappe@cetos.org 707-884-1846 (phone and fax)

Abstract

The growing clinical interest and use of soybean-based food products or extracts to increase dietary phytoestrogen intake makes the precise composition of the key biologically active ingredients of soybeans, notably genistin and daidzin of substantial medical interest. Conventional soybeans are increasingly being replaced by genetically modified varieties. We analyzed the phytoestrogen concentrations in two varieties of genetically modified herbicide tolerant soybeans and their isogenic conventional counterparts grown under similar conditions. An overall reduction in phytoestrogen levels of 12-14 percent was observed in the genetically altered soybean strains. Most of this reduction was attributable to reductions in genistin and to a lesser extent daidzin levels, which were significantly lower in modified compared to conventional soybeans in both strains. Significant sample to sample variability in these two phytoestrogens, but not glycitin, was evident in different batches of genetically altered soybeans. Given the high biological potency of isoflavones and their metabolic conversion products, these data suggest genetically modified soybeans may be less potent sources of clinically relevant phytoestrogens than their conventional precursors. These observations, if confirmed in other soybean varieties, heighten the importance of establishing baselines of expected isoflavone levels in transgenic and conventional soy products to ensure uniformity of clinical results. Disclosure of the origins and isoflavone composition of soy food products would be a valuable adjunct to clinical decision-making.

Medical interest in soy-based foods is driven by a growing number of studies indicating the phytoestrogen content of ingested soybeans can modify the pathogenesis of some hormone-dependent and hormone-independent diseases (Murkies et al, 1998; Setchell et al, 1984; Setchell & Cassidy, 1999). The popular enthusiasm for using phytoestrogens during and after menopause as a natural alternative to estrogen replacement therapy is encouraging much of the present commercialization of phytoestrogen preparations and soy extracts. Elsewhere, notably in infants fed soy-based formula (Setchell et al, 1997), concerns have been expressed about the desirability of early exposure to phytoestrogens (Robertson, 1995; Irvine et al, 1995).

Because of the widespread use of soy-based foods in medical and quasi-medical situations, knowing the amount and kind of phytoestrogens in soy products is of increasing importance. Many of these foods are derived from phytoestrogen-rich soy sources, yet the isoflavone potency of these products is unknown. In particular, there has been much controversy over the composition of newly commercialized genetically modified soybeans (Miller, 1999). The United States Department of Agriculture estimates approximately one half of the 1998 soybean crop in the United States consists of genetically modified, herbicide-tolerant soybeans, of which 33 percent are slated for export.

The phytoestrogen content of such genetically modified soybeans is thus of international interest because of the increasing global reliance on varieties of transgenic soybeans for animal and human consumption and the newly recognized physiological and pharmacological properties of isoflavones as a group (Setchell and Adlercreutz, 1988; Barnes & Peterson, 1995). The actual isoflavone concentrations in this new crop are of interest because of the growing number of clinicians who may recommend increasing dietary intake of phytoestrogens to modulate lipid oxidation (Ruiz-Larrea et al, 1997;Tikkanen et al, 1998), retard the pathogenesis of cancer, notably breast cancer ( cf Barnes et al, 1990), and reduce the risks of coronary artery disease and osteoporosis (Brandi, 1998; cf Anthony et al, 1996; and Anthony et al, 1997). Should the new transgenic soybean varieties differ significantly from conventional ones as sources of phytoestrogens, dietary recommendations would have to be modified accordingly. For this reason, we have analyzed and compared the isoflavone content of conventional and glyphosate-tolerant genetically modified soybeans for phytoestrogen content.

Concern about phytoestrogen levels in herbicide tolerant varieties which are typically over-sprayed with chemicals was precipitated by preliminary data obtained on Phaseolus species in which the authors postulated herbicide treatment may generate increased levels of phytoestrogens (Sandermann and Wellman, 1988). Preliminary phytoestrogen data on single soybean samples showed a wide range of phytoestrogen values without overt evidence of differences in isoflavone levels between conventional and genetically modified, unsprayed Roundup Ready(tm) soybeans (Padgette et al, 1996). However, no data have been published on the composition of genetically modified soybeans sprayed with glyphosate in the normal course of production.

Methods and Materials

Genetically modified soybeans were matched with conventional soybeans of the same genetic background as provided in three separate shipments by a major seed supplier (Hartz, Stuttgart Arkansas, USA). Both soybean types were grown in the Southeastern United States, had similar maturation dates, and were grown under comparable soil and climate conditions. All soybeans were dried prior to shipping to approximately 13 percent water content using standard industry techniques. The first and second batches contained H5545 Roundup Ready(tm) seed while the third contained a variety H4994, each with their conventional, isogenic counterparts.

Blinded samples of ten seeds of each variety were analyzed concurrently in pairs of conventional and glyphosate tolerant seed samples. Refrigerated seeds were pulverized and immediately extracted with cold 80% methanol. Isoflavones were isolated and measured in triplicate against an internal standard by absorption at 260 nm after separation by reverse phase, high pressure liquid chromatographic analysis as previously described (Setchell et al, 1997). Statistical analyses were performed on individual paired values using the appropriate t-test for samples with equal or unequal variance. Group means were compared after analyzing for variance using an F statistic.

Results

These data are shown in Table 1 which provides the average concentrations and 95% confidence intervals of isoflavones (in ug/gm) for a set of three analyses for each sample. In both types of soybeans, the aglycones daidzein and genistein were present in concentrations too low to permit quantitative analysis. As shown in Table 1, a majority of test pairs showed conventional soybeans to have higher isoflavone concentrations than were found in the transgenic varieties. Overall, 12 out of 21 possible analyses in the two herbicide tolerant varieties showed a significant or highly significant reduction in genistin, daidzin, or glycitin levels when compared to levels in conventional varieties (bold p values). In two instances, daidzin values were higher in transgenic than conventional soybeans.

The aggregate data of phytoestrogen levels presented in Table 2 show the two major isoflavones, genistin and daidzin, tend to be lower in genetically engineered soybeans than in their conventional counterparts. The overall reduction in concentration of total isoflavones (last column) was of borderline statistical significance in the H5545 herbicide tolerant soybeans (218.9 ug/gm or 12.2%; p= 0.052) and in the replicate H4994 variety (216.2 ug/gm or 14.4%; p= 0.058). Among the isoflavones, levels of genistein were significantly reduced in both varieties (first column): p=0.022 and p=0.036, respectively.

As reflected by the narrow 95% confidence limits shown in Table 1, individual samples analyzed in triplicate showed little within-sample variation. Additionally, some non-significant variation between sample means in conventional soybean types is evident, a finding previously reported (Eldridge and Kwolek, 1983). However, we observed appreciable variation in levels of isoflavones measured in samples taken from the isogenic batches of glyphosate tolerant soybeans. This impression was confirmed by comparing the variance of the group means measured for individual isoflavones. While conventional plants had no significant sample to sample variation, intra-group variability seen by comparing the mean variance of the conventional and genetically modified H5545 (F=3.704, p=0.05) was significant. Highly significant variance around the mean values of genetically modified soybeans was also evident for two of the three isoflavones: daidzin (F=4.76; p=.0025); and genistin (F=3.25; p=0.0199).

Discussion

The consistent reduction of genistin levels is of importance because it is enzymatically converted in the gastrointestinal tract to genistein, the most biologically potent phytoestrogen. We observed this trend towards lower genistin concentrations in both soybean varieties studied, even though these strains have different average genistin concentrations: the control H4994 strain of soybean averages about 75% of the H5545 strain genistin values (See Table 2).

The large variability seen between samples taken from batches of at least one type of genetically engineered H5545 soybean, but not its conventional counterpart, may reflect genetic variability introduced in the breeding of transgenic soybeans. Conversely, the increased between-sample variance observed in the H5545 transgenic soybean samples may reflect their greater environmental lability in responses to exogenous factors, such as herbicide oversprays, compared to conventional plants.

Overall, these data show a trend towards lower levels of isoflavones, particularly genistin, in transgenic sprayed soybeans. This alteration, if confirmed, suggests that transgenic varieties of soybeans may reduce the synthesis of specific isoflavones in the course of their development. Such a loss may simply reflect random genetic variation or drift due to the fact that the Roundup Ready(tm) varieties are not yet strictly isogenic with their matched conventional pairs. Both of the studied herbicide-tolerant varieties were derived from plants which were outcrossed to a transgenic soybean containing the glyphosate resistance gene complex and then backcrossed to the conventional line (H5545 or H4994) over approximately five generations (FS) to restore most genetic homogeneity (Spooner, 1998). The likelihood that sampling error contributed to these findings must also be considered, but the fact that our results proved reproducible in the second and third batches tested militates against this interpretation.

The observed reduction in isoflavones may be the result of an effect of the routine "over-the-top" application of glyphosate herbicide to maturing plants, a practice commonly performed usually twice during the cultivation of Roundup Ready(tm) but not the conventional varieties. The likelihood that some herbicide-related environmental factor contributes to the observed reduction is supported by the absence of isoflavone differences in non-sprayed conventional versus transgenic soybeans (Padgette et al, 1998).

The accuracy of test values within each triplicate analysis and the reproducibility of the findings of a specific reduction in specific isoflavone concentration in two different varieties of transgenic soybeans reinforces the validity of our findings. If generally true for other herbicide tolerant varieties, soybean products derived from genetically engineered food crops may be less potent dietary sources of phytoestrogens than are conventional soybeans.

Taken in aggregate, these data offer no support for the hypothesis that phyto-oestrogens are elevated in glyphosate-sprayed soybeans, but rather suggest some varieties of glyphosate tolerant soybeans may possess lower total phytoestrogen content than do their conventional counterparts. The findings of increased variability in our most intensively studied transgenic variety call into question the present tendency to equate herbicide-tolerant, transgenic with conventional soybean varieties. If our findings are confirmed by further study, those making recommendations for the medical use of soy products may wish to know the origin and/or the actual phytoestrogen levels of the soy food product used by their patients to assure consistent clinical results.

Marc Lappé, PhD, E. Britt Bailey, MA, Chandra Childress, MS, Kenneth D.R. Setchell, PhD. Center for Ethics and Toxics, Gualala, California 95445 [ML and EBB]; Children's Hospital Medical Center 333 Burnet Avenue, Cincinnati, OH 45229 [KDRS and CC].

Acknowledgments: We gratefully acknowledge the provision of soybean seed by Mr. Larry Spooner of Hartz(tm) Seed Company in Stuttgart, Arkansas. We also acknowledge the technical assistance of Mr. Noah Chalfin in performing data analysis.

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