Genistein as a potential inducer of the anti-atherogenic enzyme paraoxonase-1: studies in cultured hepatocytes in vitro and in rat liver in vivo

Abstract

A number of cardioprotective effects, including the reduced oxidation of the low-density lipoprotein (LDL) particles, have been attributed to dietary soy isoflavones. Paraoxonase 1 (PON1), an enzyme mainly synthesized in the liver, may exhibit anti-atherogenic activity by protecting LDL from oxidation. Thus, dietary and pharmacological inducers of PON1 may decrease cardiovascular disease risk. Using a luciferase reporter gene assay we screened different flavonoids for their ability to induce PON1 in Huh7 hepatocytes in culture. Genistein was the most potent flavonoid with regard to its PON1-inducing activity, followed by daidzein, luteolin, isorhamnetin and quercetin. Other flavonoids such as naringenin, cyanidin, malvidin and catechin showed only little or no PON1-inducing activity. Genistein-mediated PON1 transactivation was partly inhibited by the oestrogen-receptor antagonist fulvestrant as well as by the aryl hydrocarbon receptor antagonist 7-ketocholesterol. In contrast to genistein, the conjugated genistein metabolites genistein-7-glucuronide, genistein-7-sulfate and genistein-7,4'-disulfate were only weak inducers of PON1 transactivation. Accordingly, dietary genistein supplementation (2 g/kg diet over three weeks) in growing rats did not increase hepatic PON1 mRNA and protein levels as well as plasma PON1 activity. Thus, genistein may be a PON1 inducer in cultured hepatocytes in vitro, but not in rats in vivo.

Fig 1

Chemical structures of the test compounds as used in the PON1 reporter gene assay in stably transfected Huh7 cells.

Fig 2

Representative MRM ion chromatograms using the respective ion transition for each metabolite. (A–D) represents the chromatograms of a plasma sample from a rat showing the amounts of the selected genistein metabolites after receiving the genistein-supplemented diet (E–H) shows chromatograms of an extracted control plasma sample spiked with 50 nmol/l of each analyte. Peak * in chromatogram B was not confirmed as dihydrogenistein with the second qualifier transition. Chromatographic and mass spectrometric conditions are described in detail in the experimental section.

Fig 3

(A) Induction of PON1 transactivation by different flavonoids in stably transfected Huh7 liver cells. (B) The chemical structure of genistein and dose-dependent effect of genistein on PON1 transactivation in stably transfected Huh7 liver cells. (C) The chemical structure of the genistein-metabolite genistein-7-glucuronide (G7-MGluc) and dose-dependent effect of G7-MGluc on PON1 transactivation in stably transfected Huh7 liver cells. *statistical significant differences at P ≤ 0.05; anova. (D) The chemical structure of the genistein-metabolite genistein-4′-sulfate (G4′-MSulf) and dose-dependent effect of G4′-MSulf on PON1 transactivation in stably transfected Huh7 liver cells. (E) The chemical structure of the genistein metabolite genistein-7,4′-disulfate (G7,4′-DSulf) and dose-dependent effect of G7,4′-DSulf on PON1 transactivation in stably transfected Huh7 liver cells. Reporter gene data are mean with S.E.M. of three experiments performed in triplicate.

Fig 4

(A) Oestrogen response elements (ERE) in alternative promoter sequences of the PON1 gene. ERE binding sites are depicted in alternative promoter sequences of PON1 (GXP_116467, GXP_116464, GXP_116678, GXP_2253723) relative to transcription start site (red arrow). (B) Western blotting of oestrogen receptors α and β in Huh7 whole cell lysates indicates presence of both oestrogen-receptor variants in Huh7 cells. Actin served as loading control. (C) Western blotting of aryl hydrocarbon receptor (AhR) in Huh7 whole cell lysates indicates presence of AhR in Huh7 cells. Actin served as loading control. (D) Aryl hydrocarbon receptor-binding sites in alternative promoter sequences of the PON1 gene. ERE-binding sites are depicted in alternative promoter sequences of PON1 (GXP_116467, GXP_116464, GXP_116678, GXP_2253723) relative to transcription start site (red arrow).

Fig 5

(A) Inhibition of the genistein-mediated PON1 transactivation by the oestrogen-receptor antagonist fulvestrant in stably transfected Huh7 liver cells. Cells were treated for 48 hrs. Data are mean + S.E.M. of three experiments performed in triplicates. (B) Inhibition of the genistein-mediated PON1 transactivation by the aryl hydrocarbon receptor antagonist 7-ketocholesterol (7-kc) in stably transfected Huh7 liver cells. Cells were treated for 48 hrs. Data are mean + S.E.M. of three experiments performed in triplicates. *Mean values were significantly different from the genistein treatment without 7-ketocholesterol (P ≤ 0.05), Mann–Whitney U-test.

Fig 6

Effect of a 3-week supplementation with dietary genistein (2 g/kg) on PON1 mRNA (A) and protein levels (B) as well as plasma PON1 activity (C) in male Wistar Unilever rats compared with control-fed rats. ** indicate statistical significant differences at P ≤ 0.01, t-test.