Evaluation of the hazelnuts as functional food: molecular effects of long maceration raw kernel extract in HepG2 human hepatocarcinoma cell line

Background: Several clinical studies have reported the beneficial effects of a hazelnut-enriched diet in preventing the development of cardiovascular diseases [Sabate et al., 2003]. In this context, we recently carried out a case study on healthy volunteers and demonstrated that the blood lipid profile was significantly improved in subjects undergoing a diet supplemented with 40 grams of fresh hazelnut for 6 weeks [Santi et al., 2017].  Objective: In order to identify the mechanisms responsible for the effect observed in our case study, we investigate the cellular and molecular effects triggered by the administration of a galenic extract of fresh hazelnut on the HepG2 human hepato-carcinoma cell line. Methods: The hazelnuts extract is a hydro alcoholic solution of 20 g of fresh hazelnut (Corylus avellana, L.).  It was left to macerated in the dark for 30 days, then filtered, and analyzed by liquid chromatography-mass spectrometry (LC-MS) for metabolomic characterization. The pH, the total poliphenols and the antioxidant activity of the solution was furthermore measured by spectrophotometric assay using theTrolox method and compared to the raw product. The HepG2 cell line (which is routinely used for liver in vitro studies as it keeps unaltered most metabolic hepatic functions) was treated for 24h and 72h with the galenic extract at concentrations ranging from 0.004 to 4 mg/ml. At the indicated times, cells were counted, assessed for viability by trypan blue exclusion, and the cellular toxicity was specifically evaluated by the Cell Titer Fluor Viability Assay (Promega). Total RNA was extracted by TRIZOL (Invitrogen), retro-transcribed into cDNA, and the expression of the Low-Density Lipoprotein Receptor (LDLr), apolipoproteins (APOs) family genes, DNA methyl transferases DNMT-1 and -3 was analyzed with real time PCR (qRT-PCR) (Ecoillumina) by normalizing to GAPDH, and tubulin and r18s levels using the ΔΔCT method. The expression of the microRNA-122 was also analyzed in qRT-PCR by Exiqon technology through normalization with the RNU1A1 small nuclear RNA. Results: The metabolomic characterization identified around 80 functional primary and secondary biomolecules, the main metabolic class was represented by the phenylpropanoids (39%) followed by lipids (27%), phenolic acids (14%), vitamins (12%), and sugars (8%). The galenic solution also exhibited the greatest antioxidant activity compared to raw hazelnut in terms of μmol Trolox eq/100g (4.92 vs 3.26 p=0,001). By administrating HepG2 cells with the galenic solution, we demonstrated the lack of any liver toxicity within the dose range chosen. Interestingly, there was a dose-response increment of LDLr and ApoA1, ApoE and ApoB100 expression at 24h of treatment, which was stably maintained up to 72h of continuous exposure to the hazelnut solution. In terms of epigenetic changes, no detectable levels of the liver specific microRNA-122 was observed in the HepG2 cells, nor in response to the hazelnut administration. The expression of the DNMTs genes was indeed stimulated in response to the galenic solution administration, with the highest induction observed at the lowest doses of hazelnut concentrations. The analysis of the methylation level of global genomic DNA as well as at promoter level (LDLr and Apo genes), carried out by DNA bisulfite conversion and pyrosequncing, is currently undergoing. Conclusion: We hypothesized a strong link between the high lipid content of the galenic extract, and the macroscopic effects on the HepG2 cells. In addition, the increased expression levels of the genes regulating the lipid metabolism is in line with the lipid profile improvement reported in our human case study, and suggest that hazelnut may impact on blood parameters by affecting key regulatory genes at liver level. The modulation of the DNMTs expression upon hazelnuts administration indicate a significant involvement of the epigenetic dynamics by DNA methylation in response to diet supplementation.  These findings provide clues for further multi-level analyses to disclose the specific involvement of such epigenetic modulator on gene expression that may account for the reported effects on lipid profile.  Overall, our data demonstrates the significant antioxidant properties of the hazelnuts and opens a new scenario on the ability of such diet supplements to affect the transcription and epigenetic profile of target tissues. In conclusion, these data support the hazelnut as a promising functional food in the prevention of those pathologic states that correlate with an altered blood lipid profile. Keywords:  hazelnut, functional food, liver, lipid metabolism, epigenetics. How to cite this abstract:  Santi C., Benassi B., Diretto G., De Murtas O., Pacchierotti F., Bacchetta L.; FFC’s 22 nd International Conference; Boston, MA, USA; Organized by FFC and BIDMC/ Harvard Medical School Teaching Hospital; Volume 1; Supplement 1: 77-79