Evaluation of mutagenicity, genotoxicity and alterations in the gene expression profile induced by the synephrine in human cells in vitro

Author(s)

Diego Luís Ribeiro

• Document type: Doctoral Thesis
• Press: Ribeirão Preto.
• Institution: Universidade de São Paulo (USP). Faculdade de Medicina de Ribeirão Preto (PCARP/BC)
• Defense date: 2018-04-06

Examining board members

Lusania Maria Greggi Antunes; Farah Maria Drumond Chequer; Ilce Mara de Syllos Colus; Tiago Campos Pereira; Catarina Satie Takahashi

• Advisor: Lusania Maria Greggi Antunes; Gustavo Rafael Mazzaron Barcelos

Abstract

Currently, obesity is one of the biggest global health problems because it increases the risk of developing diseases like cancer. As an alternative to weight loss, thermogenic supplements containing the proto-alkaloid synephrine are widely used to stimulate the burning of calories. Doubts have arisen about the effects of synephrine since it has similarities with ephedrine and amphetamines. There is still a gap in the literature on the effects of synephrine on the induction of DNA damage and modulation of gene expression in human cells. The aim of this investigation was to determine the effects of synephrine treatments on DNA damage, redox status and alterations in gene expression in liver, gastric and intestinal human cell lines in vitro. As model of human cells were used HepG2 (hepatocellular carcinoma), GAS (gastric epithelium non-tumor) and Caco-2 (colorectal adenocarcinoma) cells. Initially, cytotoxicity assays (MTT and Neutral Red) were performed with ten concentrations (25-5000 µM). Afterwards, three concentrations (2, 20 and 200 µM) were selected to obtain results on cell proliferation (protein content and flow cytometry), genotoxicity (comet), mutagenicity (micronucleus), generation of reactive species (RS) (H2DCFDA), and changes in the redox state by quantification of GSH, CAT, mitochondrial SOD and GPx enzymes and the peroxidation product malondialdehyde (MDA). Finally, effects of synephrine on gene expression (by RT-qPCR) in pathways of DNA damage and energetic metabolism were evaluated. Synephrine induced RS in hepatic (HepG2) and intestinal (Caco-2) cells; in addition, in GAS increased CAT and GSH levels, in Caco-2 induced the GSH levels and, mainly in hepatic cells (HepG2) were observed increases in enzymatic levels of GSH, GPx and generation of MDA. In gene expression experiments, 20 and 200 µM increased the transcription of the MAPK1 and JUN genes in HepG2, GAS and Caco-2, evidencing that treatment with synephrine stimulate the expression of AMPK (Adenosine Monophosphate Protein Kinases Cytosolic Activating) pathway, responsible by energetic metabolism of lipids and glucose. Specifically, in HepG2 cells synephrine induced expression of TNF (pro-inflammatory cytokine) and decreased of LIPE (lipid hydrolysis) and AKT1 (proliferation) gene expression. In GAS, synephrine decreased TNF expression and increased expression of the genes ADCY3 (catalytic activity) and AKT1. Similarly, in Caco-2, AKT1 and the genes PRKACA and GNAS (catabolism) also increased expression. Finally, only in Caco-2, 2 µM increased the transcript levels of ATM, ATR and CHEK1 (DNA damage). Despite these results, no effects on viability, proliferation, cycle kinetics and DNA damage were observed. In conclusion, this work demonstrated that synephrine did not cause cytotoxicity, genotoxic and mutagenic effects in liver, gastric and intestinal human cells in vitro. However, as well as amphetamines, synephrine has been shown to stimulate significantly the expression of energetic metabolism genes and cause pro-oxidative effects mainly in liver cells. Therefore, in order to represent the absence of hazard of synephrine, experiments with chronic exposures using in vivo studies should be performed for affirmative conclusions to thermogenic users. (AU)

• FAPESP's process: 14/20344-9 - Mutagenicity, genotoxicity and changes in gene expression profile evaluation of synephrine dietary supplement in human cells in vitro
• Grantee: Diego Luis Ribeiro
• Support Opportunities: Scholarships in Brazil - Doctorate