Elsevier

Reproductive Toxicology

Volume 15, Issue 6, November–December 2001, Pages 647-663
Reproductive Toxicology

Effects of dietary genistein exposure during development on male and female CD (Sprague-Dawley) rats

https://doi.org/10.1016/S0890-6238(01)00177-0Get rights and content

Abstract

Genistein is a naturally occurring isoflavone that interacts with estrogen receptors and multiple other molecular targets. Human exposure to genistein is predominantly through consumption of soy products, including soy-based infant formula and dietary supplements. A dose range-finding study was conducted as a prelude to a multigeneration bioassay to assess potential toxicities associated with genistein consumption. Genistein was administered in a soy- and alfalfa-free diet at 0, 5, 25, 100, 250, 625, or 1250 ppm to pregnant dams starting on Gestation day 7 and continuing throughout pregnancy. Dietary exposure of the dams continued through lactation, and pups were maintained on the same dosed feed as their mother after weaning until sacrifice at Postnatal day 50. Body weight and feed consumption of the treated dams prior to parturition showed a decreasing trend with a significant reduction at the highest dose. Litter birth weight was depressed in the 1250 ppm dose group, and pups of both sexes in that dose group had significantly decreased body weights relative to controls at the time of sacrifice. The most pronounced organ weight effects in the pups were decreased ventral prostate weight in males at the 1250 ppm dose and a trend toward higher pituitary gland to body weight ratios in both sexes. Histopathologic examination of female pups revealed ductal/alveolar hyperplasia of the mammary glands at 250 to 1250 ppm. Ductal/alveolar hyperplasia and hypertrophy also occurred in males, with significant effects seen at 25 ppm and above. Abnormal cellular maturation in the vagina was observed at 625 and 1250 ppm, and abnormal ovarian antral follicles were observed at 1250 ppm. In males, aberrant or delayed spermatogenesis in the seminiferous tubules relative to controls was observed at 1250 ppm. There was a deficit of sperm in the epididymis at 625 and 1250 ppm relative to controls, although testicular spermatid head counts and epididymal spermatozoa counts did not show significant differences from controls at these doses. Both sexes showed an increase in the incidence and/or severity of renal tubal mineralization at doses of 250 ppm and above. Dietary genistein thus produced effects in multiple estrogen-sensitive tissues in males and females that are generally consistent with its estrogenic activity. These effects occurred within exposure ranges achievable in humans.

Introduction

The consumption of diets with high levels of soy has been related to multiple beneficial effects, including chemopreventive activities against various cancers and alleviation of some of the adverse consequences of menopause [1], [2], [3], [4], [5], [6], [7], [8]. Diets high in soy contain multiple agents that may contribute to these effects, and consumption of these diets is also associated with lower calorie and fat intake. Nonetheless, much research attention has focused on the isoflavones, and particularly genistein, as the active components contributing to (or responsible for) the beneficial effects of soy. This is due to the demonstrated interaction of soy isoflavones, particularly genistein, with estrogen receptors, effects on hormone synthesis and metabolism and sex hormone binding proteins, and genistein’s ability to modulate multiple enzymes involved in growth regulation, including tyrosine kinases and topoisomerases. These activities have been extensively reviewed (see above references). The association of diets containing soy with lower rates of many common Western health problems has led to the development of concentrated isoflavone-containing plant extracts for use as dietary supplements that are beginning to be evaluated in clinical trials [e.g. [9], [10]]. In addition, soy-based infant formulas have been available for decades, and infants consuming soy formula have been shown to have levels of circulating isoflavones as high as 5 to 10 μM [11].

Research assessing the potential adverse effects associated with isoflavone consumption is primarily directed toward defining any potential risk from exposure to a range of doses of isoflavones during different life stages. Developmental stages are of particular concern because of the demonstrated adverse consequences of exposure to hormonally active agents, such as diethylstilbestrol, during development [12], [13], [14], although potential adverse stimulatory effects of genistein on reproductive and breast tissues of postmenopausal women also require particular attention [15], [16], [17]. In addition, levels of human exposure to these dietary endocrine modulators are generally much greater than exposure to synthetic chemicals with hormonal activity; thus, a better understanding of the effects of these compounds on hormone-sensitive endpoints is important in evaluating the hypothesis that hormonally active agents are adversely impacting human and wildlife populations. Finally, because of the widespread exposure of humans to significant doses of soy isoflavones at various stages of development, the data obtained from animal models can be evaluated for its relevance to human risk assessment more readily than is the case for other endocrine active chemicals with limited human exposure.

Adverse effects of soy-containing foods and soy components on reproductive processes of animals have been reported [18], [19], [20], [21], and human studies have indicated the consumption of soy products can have hormonal effects in women [22], [23], [24], [25], [26], [27], [28], [29]. It has been suggested, based on studies in ovariectomized animals, that the beneficial effects of soy and its component isoflavones on the cardiovascular system and bone occur at doses that do not adversely affect the reproductive tract [30], [31]. In addition, inhibition of chemically induced mammary cancer in rats has been reported at doses that did not produce adverse effects on reproductive tissues [32], [33], [34]. However, given the potential range of effects of soy and its components and the potential for increased consumption of soy-containing products, it is important to conduct comprehensive toxicologic evaluations of these agents to better understand potential adverse effects that could result from their use. A growing body of toxicologic data on genistein in rodents is accumulating that confirms the importance of dose, timing of dose, target organ, route and context of administration (i.e. pure aglycone versus soy or soy extract), and species on the resulting effects. The purpose of this study was to assess potential toxicities associated with genistein consumption and to select appropriate doses for evaluation in a multigeneration study. Doses for the present study were selected to cover isoflavone exposure levels in humans as well as doses, on a mg per kg body weight basis, that have been reported to have effects on target organs in animals, and a comprehensive battery of endpoints was evaluated.

Section snippets

Animals and husbandry

Female CD (Sprague-Dawley) rats (NCTR Strain Code 23), 70–80 days old, were shifted from the standard NIH-31 pellet diet to a soy- and alfalfa-free meal diet (see below) one week prior to breeding to untreated males. Ten vaginal plug-positive untreated females were randomly assigned on gestation day 7 (GD7, plug date = day 0) to each dose group to ensure that five litters would be obtained from each group. The plug-positive females were housed individually in standard polycarbonate cages with

Genistein intake

The approximate range of doses of genistein ingested by the animals over various phases of the study as determined from the mean feed consumption and body weight data are shown in Table 1. Feed consumption was assessed over various time periods, daily for dams during pregnancy and weekly for dams between litter delivery and weaning and for pups after weaning, and reflects both consumption and spillage.

Effects of dietary genistein on body weight and feed consumption of dams during pregnancy and after delivery

Body weights of the 1250 ppm dose group were significantly less than controls on GD 20 and

Discussion

The doses of genistein ingested by the rats in this study (Table 1) spanned a range that covered estimated human isoflavone exposures resulting from consumption of soy foods in adults (approximately 1 mg/kg/day) and of soy infant formula (6 to 9 mg/kg/day) [11] up to near 200 mg/kg/day. It is important to note that the actual internal dose of genistein, as assessed by serum levels, achieved over the dose range tested in the present study, even at the high end of the dose range, are within the

Acknowledgements

This work was supported by Interagency Agreement 224–93-0001 between the National Institute for Environmental Health Sciences and the United States Food and Drug Administration. We thank Dr. Julius Thigpen of NIEHS for conducting mouse uterotrophic assays on the 5K96 diet. We also appreciate the considerable efforts of the experimental liaison and biostatistical support groups of ROW Sciences, the animal care and diet preparation staffs of Bionetics, the staff of Pathology Associates

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