An investigation into the biochemical, molecular and epigenetic effects of fumonisin B1 in liver (HEPG2) cells.

Abstract

Fumonisins are carcinogenic mycotoxins that occur world wide in maize and maizebased products intended for human consumption. Consumption of fumonisincontaminated maize as a staple diet has been associated with oesophageal and liver cancer in South Africa and China. Fumonisin B1 (FB1) inhibits sphingolipid biosynthesis and has been implicated in cancer promoting activity in animals and humans. FB1 disrupts DNA methylation and induces chromatin modifications in human hepatoma (HepG2) cells. In this study FB1 (IC50=200μM) altered liver enzyme expression of DNA methyltransferases and demethylases. DNA methyltransferase activities of DNMT1, 3a and 3b were significantly decreased, whilst both DNA methylase (MBD2) activity and expression was significantly up-regulated resulting in global DNA hypomethylation. In addition the histone demethylases, KDM5B and KDM5C, expression was increased. FACS data confirmed FB1 significantly increased global DNA hypomethylation – a process that causes chromatin instability. Next the effect of FB1 on miRNA expression was evaluated; FB1 significantly down-regulated (11 fold) expression of miR-27b. MiR-27b modulates expression of human cytochrome P450 (CYP1B1) that catalyzes the metabolic activation of many procarcinogens. In order to directly assess the effect of miR-27b on CYP1B1 mRNA levels, liver cells were transfected with the mimic to miR-27b. CYP1B1 mRNA and protein expression was significantly up-regulated by 1.8- fold and 2.6- fold respectively. CYP1B1 is post-transcriptionally regulated by miR-27b suggesting that FB1- induced modulation of miR-27b in hepatic cells may be an additional mode of hepatic neoplastic transformation. Finally, the effect of FB1 on the apoptotic pathway in HepG2 cells was investigated using an mRNA expression array panel of pro- and anti- apoptotic molecules. FB1 significantly increased an AIP family member - BIRC 8/ILP-2 (8-fold) in an apoptosis array. In addition, ILP2 protein expression was increased (2.3-fold) with a corresponding decrease in Smac/DIABLO protein levels (1.7-fold). Further analysis showed an FB1 (0μM, 50μM, 100μM, 200μM) dosedependent increase in BIRC-8/ILP-2 mRNA and protein expression in HepG2 cells. This data suggests that FB1 modulates apoptosis in a complex dose-dependent regulation of pro- and anti-apoptotic molecules – and it is not a matter of simply switching on or off. In conclusion, the data shows that FB1 possess epigenetic properties by inducing global DNA hypomethylation, modulating miRNA expression, and increasing expression of the AIP protein family (BIRC8/ILP-2) that may lead to liver tumourigenesis.