Plasma bioavailability and changes in PBMC gene expression after treatment of ovariectomized rats with a commercial soy supplement

Abstract

The health effects of soy supplementation in (post)menopausal women are still a controversial issue. The aim of the present study was to establish the effect of the soy isoflavones (SIF) present in a commercially available supplement on ovariectomized rats and to investigate whether these rats would provide an adequate model to predict effects of SIF in (post)menopausal women. Two dose levels (i.e. 2 and 20 mg/kg b.w.) were used to characterize plasma bioavailability, urinary and fecal concentrations of SIF and changes in gene expression in peripheral blood mononuclear cells (PBMC). Animals were dosed at 0 and 48 h and sacrificed 4 h after the last dose. A clear dose dependent increase of SIF concentrations in plasma, urine and feces was observed, together with a strong correlation in changes in gene expression between the two dose groups. All estrogen responsive genes and related biological pathways (BPs) that were affected by the SIF treatment were regulated in both dose groups in the same direction and indicate beneficial effects. However, in general no correlation was found between the changes in gene expression in rat PBMC with those in PBMC of (post)menopausal women exposed to a comparable dose of the same supplement. The outcome of this short-term study in rats indicates that the rat might not be a suitable model to predict effects of SIF in humans. Although the relative exposure period in this rat study is comparable with that of the human study, longer repetitive administration of rats to SIF may be required to draw a final conclusion on the suitability of the rat a model to predict effects of SIF in humans.

Keywords: BPs, biological pathways; Bioavailability; DMSO, dimethyl sulfoxide; Dose effect; E2, estradiol; ECM, extracellular matrix; EREs, estrogen-responsive elements; ERs, estrogen receptors; GSEA, gene set enrichment analysis; Gene expression; HD, high dose; HPLC, high performance liquid chromatography; KEGG, kyoto encyclopedia of genes and genomes; LD, low dose; MDS, multidimensional scaling; NCBI, National Center for Biotechnology Information; PBMC, peripheral blood mononuclear cells; SIF, soy isoflavones; Soy supplementation; Species differences; UPC, universal expression code.

Fig. 1

Plasma curves of isoflavones in F344 ovariectomized rats after a single oral gavage dose (2 and 20 mg/kg b.w.) of a commercial soy supplement. Only daidzein and equol could be detected (the limit of detection for all SIF was 0.01 μg/ml, and the limit of quantification was 0.02 μg/ml). Data are expressed as mean ± SD. Sample size 5 rats/group. LD (▴) and HD (♦) stand for low and high dose group, respectively. A significant effect in LD and HD at specific time points is denoted by * (p < 0.01). The inserted graph shows the plasma curve of daidzein during the first 2 h.

Fig. 2

Amount of isoflavones in urine (a) and feces (b) of F344 female ovariectomized rats over a 24 h time period after administration of a single oral dose (2 and 20 mg/kg bw SIF) of a commercial soy supplement. Data are expressed as mean ± SD. Sample size 5 rats/group. LD and HD stand for low and high dose, respectively. Significant effects between LD and HD groups compared to control at specific time points are denoted * (p < 0.01) and ** (p < 0.001).

Fig. 3

Flow chart of microarray analysis.

Fig. 4

Multidimensional scaling (MDS) plot off all arrays. The plot was generated using the top 500 most affected genes. The distances in both dimensions are arbitrary units where LH, HD and cont. stands for low dose, high dose and control rats, respectively.

Fig. 5

Log (base 2) fold change (FC) plot of LD vs. HD groups compared to controls. The log2 FC plots were prepared using data of all 7650 genes.

Fig. 6

Rank–rank scattered plot for significantly changes in gene expression among the LD and the HD group relative to the controls (a), and for the LD group compared to (post)menopause women (b). A similar picture was found for HD rats vs. (post)menopause women (data not shown). Out of 7650 filtered genes (see Fig. 3) 3575 genes were significantly changed (p < 0.05) in both LD and HD groups; while out of 7650 filtered genes, only 69 genes were significantly changed (p < 0.05) in both rat and homologous human PBMC. These 3575 and 69 genes were used to build the scattered plot. Each dot represents the t-value of a single gene. Corresponding human homologous genes were retrieved from the study with (post)menopausal women taking the same supplement, in a similar dose as the LD rats . Genes significantly changed in the same direction in both treatments are in Cartesian quadrants I and III, while genes significantly changed in opposite directions are in quadrants II and IV. The density of the dots visualize similar trends in regulation among the two different groups.