Research SectionSafety evaluation of phytosterol esters. Part 1. Assessment of oestrogenicity using a combination of in vivo and in vitro assays
Introduction
Phytosterols, or plant sterols, are natural constituents of the human diet and are commonly found as minor constituents (0.1–0.5%, w/w) of edible vegetable oils (Kochhar, 1983). They are structurally related to cholesterol, but differ in their side-chain configuration (Fig. 1). There is a wide variety of phytosterol structures but the most frequent phytosterols found in nature are β-sitosterol, campesterol and stigmasterol which occur in the free form or esterified to free fatty acids, sugar moieties or phenolic acids. Dietary intake of phytosterols in northern European countries has been estimated to be 200–300 mg/day (Morton et al., 1995), with intakes in vegetarians and Japanese estimated to be higher (300–450 mg/day) (Nair et al., 1984; Nakashima et al., 1981).
Phytosterols esterified with fatty acids from sunflower oil, referred to as phytosterol esters (PE), are being investigated for use as a novel food ingredient, primarily for use in margarine and spreads as a functional component with blood cholesterol-lowering properties. Esterification is important to increase their solubility in the oil phase of the spread.
A number of reports in the literature have suggested that phytosterols may have effects on the reproductive system, and in particular that they possess oestrogenic activity (Elghamry and Hensel, 1969; Malini and Vanithakumari, 1993; Mellanen et al., 1996; Rosenblum et al., 1993; Samannoudy et al., 1980). However, many of these studies were not designed in a way appropriate for assessing the potential hazard of materials intended for oral consumption, and in addition, crude plant extracts were often used and purity of individual phytosterols not specified.
Therefore, as part of an extensive programme of safety evaluation studies, the oestrogenic potential of phytosterols (a well characterized mixture of β-sitosterol, campesterol and stigmasterol) was assessed using a combination of in vitro and in vivo assays. The first assay, competitive binding with the oestrogen receptor (ER), uses a cell-free system to determine the extent to which the compound binds to the ER, as reflected by its ability to displace 3H-oestradiol from the ER (Ireland et al., 1980). Secondly, the transcriptional activation of oestrogen responsive genes has been examined in an oestrogen-inducible yeast screen (Routledge and Sumpter, 1996). This assay employs a yeast strain (Saccharomyces cerevisiae) transfected with human ER and an oestrogen-responsive element (ERE) linked to the reporter gene lac-Z (encoding the enzyme β-galactosidase). Thus, in the presence of an oestrogenic compound, β-galactosidase is synthesized and secreted into the medium, where it is detected as a colorimetric change. The third assay was used to determine the effects of phytosterols on an oestrogen-responsive tissue in an intact animal. The immature rat uterotrophic assay (Wakeling et al., 1991), one of the most widely used methods to detect oestrogenicity, was used to determine whether oral exposure to phytosterols led to increased uterine weight. Therefore, by providing information on three levels of hormonal activity, an informative profile of oestrogenic potential could be obtained.
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Chemicals
β-oestradiol (E2), diethylstilbestrol (DES), cholesterol and cholesteryl palmitate were obtained from Sigma (Poole, Dorset, UK). [2,4,5,6-3H]oestradiol (3H-E2) was obtained from Amersham (Little Chalfont, Buckinghamshire, UK). β-Sitosterol (purity by gas chromatography >99%) was obtained from Sigma. Coumestrol [purity by gas chromatography–mass spectrometry (GC–MS) >99%] was obtained from Apin Chemicals Ltd (Abingdon, Oxfordshire, UK). Phytosterols, analysed by GC–MS as a mixture of β
Results
Results from the in vitro competitive ER binding assay, the recombinant yeast screen and the in vivo uterotrophic assay are presented. β-Estradiol was included as a positive control in all the studies to monitor and ensure the performance of the test systems and to provide reference data against which to compare the results of the other test substances. In addition coumestrol, a coumestan with known oestrogenic activity (Gaido et al., 1996; Markaverich et al., 1995; Odum et al., 1997; Whitten
Discussion
Reports in the literature have suggested that phytosterols may have effects on the reproductive system and in particular that they possess oestrogenic activity (Elghamry and Hensel, 1969; Malini and Vanithakumari 1993; Mellanen et al., 1996; Rosenblum et al., 1993; Samannoudy et al., 1980). As part of an extensive programme of safety evaluation studies, the oestrogenic potential of phytosterols was assessed using a combination of in vitro and in vivo assays.
A study by Rosenblum et al. (1993)
Acknowledgements
The authors would like to thank Dr Ed Routledge for performing some of the yeast assays.
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