Bread crust extract is a novel activator of aryl hydrocarbon receptor and modulator of NRF2 and NFκB in HepG2 and HCT 116 cells

Abstract

The Maillard reaction describes the non-enzymatic formation of advanced glycation end products (AGEs), e.g., during thermal food processing. Studies on the mode of action and health implications of food-derived AGEs are often contradictory and lack information on active components. We use bread crust extract (BCE) as a model for an AGE-rich diet. Despite the identified AGEs and known activated signaling pathways, it is still unclear which receptors can exert the various effects described for BCE. This study investigates whether BCE can induce the aryl hydrocarbon receptor (AHR), the downstream NRF2 and NFκB signaling pathways and if this activation can be attributed to individual, free AGEs or AHR-agonists present in BCE. HepG2 reporter cell results showed activation of AHR and NRF2 but not NFκB by BCE. However, the tested free AGEs did not show an activation. Known AHR-(pro-)agonists kynurenine (Kyn) and benzo[a]pyrene (BaP), both present in BCE, activated the reporter to a similar extent as BCE with distinct differences in target gene induction of CYP1A1, interleukin-8, heme oxygenase 1 and Manganese-superoxide dismutase. Furthermore, CYP1A1 ethoxyresorufin-O-deethylase enzymatic activity was also induced and could be modulated by AHR and NRF2-inhibition. In contrast, in HCT 116 pTRAF reporter cells, BCE activated AHR, NFκB and NRF2 and induced CYP1A1. We conclude that BCE contains potent AHR activators that influence cellular signaling activities. AHR most likely concerts cell line dependent NRF2 and NFκB-activation. The BCE effects are probably attributable to an interplay of AHR-agonists and AGEs.

Keywords: Advanced glycation end products; Aryl hydrocarbon receptor; Bread crust extract; Maillard reaction; NFκB; NRF2.

Graphical abstract

Fig. 1

BCE-mediated luciferase signal induction in HepG2 reporter cells. Data are presented as mean fold-change above media controls ±SD. p-values ∗ <0.05, ∗∗ <0.01, ∗∗∗ <0.001. A: AHR-dependent Luciferase signals are induced in a dose-dependent manner by BCE after 24 h and to a greater extent after 48 h of incubation. N = 3, n = 3, Pos. ctrl.: 1.8 μM FICZ. B: NRF2- but not NFκB-HepG2-reporter cells showed dose-dependent induction of luciferase signals by BCE. N = 7, n = 3. Pos. ctrl.: 5 μM tBHQ, 20 ng/ml TNFα. FC: fold change, Pos. ctrl.: positive control(s).

Fig. 2

Induction of luciferase expression in HepG2 reporter cells by known AHR-(pro-)agonists. N = 5–8, n = 3. p-values ∗ <0.05, ∗∗ <0.01, ∗∗∗ <0.001. A: Kyn is able to significantly and dose-dependently induce AHR-dependent and to a lesser extend NRF2-dependent luciferase expression at 24 h of incubation. B: BaP significantly induces AHR from 62.5 nM, NRF2 from 15.6 nM and at 31.3 nM also NFκB-dependent luciferase expression in HepG2-reporter cells. FC: fold change.

Fig. 3

Target gene induction in HepG2 cells upon stimulation with BCE, Kyn and BaP. N = 3, n = 3. P-values ∗ <0.05, ∗∗ <0.01. A: BCE strongly induces the AHR-target gene CYP1A1, Heme oxygenase 1 (HMOX1) and interleukin 8 (IL-8) but not Manganese superoxide dismutase (Mn-SOD). B: Target gene activation of AHR-(pro-)agonists BaP, Kyn and FICZ.

Fig. 4

AHR activation and induction of NRF2 and NFκB reporter-fluorescence by BCE, Kyn and BaP in HCT 116 reporter cells. A: Significant NRF2-dependent mCherry induction was reported for all concentrations of BCE, for Kyn (N = 3) and for the positive control, 2 μM auranofin (N = 4) (AF) after 24h of stimulation. N = 3–6 for controls. P-values against solvent controls (PBS/DMSO) ∗ < 0.05, ∗∗∗ < 0.001. B: Induction of NFκB reporter fluorescence (TFP) was seen in 5 %–10 % BCE and the controls FICZ and 10 ng/ml TNFα (all N = 3) after 24 h incubation. N = 3–6 for DMSO and medium-controls. P-values: ∗∗∗ <0.001. C&D: All tested inducers showed significantly increased CYP1A1 expression as a result of AHR-activation. Only BCE induced HMOX1 and interleukin IL-8 in a dose-dependently. Mn-SOD was not induced. N = 3, n = 3. E–H: AHR-degradation half-life and dynamics after activation were analyzed by anti-AHR Western blot for (E) 5 % BCE, (F) 150 μM Kyn, (G) 500 nM BaP, and (H) the positive control FICZ at 2 h, 4 h, 6 h, 8 h and 24 h of incubation. All substances showed varying extents of AHR-degradation, with the longest half-life (t_1/2) in BCE. N = 3. P-values from one-sample t-tests, ∗ <0.05, ∗∗ <0.01.

Fig. 5

Induction of CYP1A1 activity by BCE, Kyn and BaP in HepG2 and HCT 116 cells determined by the EROD assay after 24 h of induction. A: HepG2 cells, N = 6, n = 3. B: HCT 116 cells, N = 4, n = 3. The AHR antagonist CH-223191(1 μM) and NRF2-inhibitor Brusatol (30 nM) were used in HepG2-cells to test for AHR-specificity and NRF2 interplay. Denotion of p-values from unpaired t-tests against medium controls ∗ <0.05, ∗∗ <0.01, ∗∗∗ <0.001, p-values from unpaired t-tests of inhibition against effects § < 0.05, §§ < 0.01, §§§ < 0.001.