Aflatoxin B1 induces persistent epigenomic effects in primary human hepatocytes associated with hepatocellular carcinoma
Introduction
Exposure to the highly potent carcinogenic and hepatotoxic mycotoxin aflatoxin B1 (AFB1) through the food chain presents a significant risk factor for the development of hepatocellular carcinoma (HCC) worldwide (Liu and Wu, 2010, Wild and Gong, 2010). Especially populations in regions such as sub-Saharan Africa, Southeast Asia, and China are at risk of developing HCC due to the combination of high hepatitis B virus prevalence and largely uncontrolled dietary AFB1 exposure (Liu and Wu, 2010). Of the 550,000–600,000 new HCC cases worldwide each year, about 25,000–155,000 may be attributable to AFB1 exposure (Liu and Wu, 2010). Therefore, AFB1 exposure may have a causative role in 4.6–28.2% of all global HCC cases (Liu and Wu, 2010). Against this background, a better understanding of the AFB1 mechanism-of-action may be of value for underlining policy measures aiming at reducing exposure to this foodborne carcinogen.
Specific cytochrome P450 enzymes in the liver metabolize AFB1 into the highly reactive AFB1-8,9-epoxide, which may then bind to proteins and cause acute toxicity (aflatoxicosis) or to DNA to initiate mutations that over time increase the risk of HCC (Groopman et al., 2008). Especially, the transversion mutation (G → T) in codon 249 of the p53 tumor suppressor gene has been associated with AFB1 exposure (Soini et al., 1996). A mutation in this gene may cause silencing of its tumor suppression function and allow uncontrolled cell proliferation.
Besides inducing genetic effects, AFB1 exposure also has been reported to affect the epigenome (Zhang et al., 2002, Zhang et al., 2003, Zhang et al., 2006, Zhang et al., 2012).
“Epigenetic memory” has been proposed as an indicator of prior exposure (Mirbahai and Chipman, 2014) to for example AFB1, ultimately leading to the development of HCC. Epigenetic alterations induced by environmental stressors, such as AFB1, including changes of the normal DNA methylation pattern and microRNA expression alterations, can create a persistent memory of the received signal. It is hypothesized that each class of toxicants (with a specific “mode of action”) can induce class-specific alterations in the normal pattern of DNA methylation or microRNA expression pattern (also called an “epigenetic foot-print”) (Goodman et al., 2010, Koufaris et al., 2012, Legler, 2010, Mirbahai and Chipman, 2014, Moggs et al., 2004). These changes will thereupon induce alterations in the gene expression profile, which may promote changes in an organism’s traits either immediately or at a later stage (e.g. development of HCC) (Izzotti and Pulliero, 2014, Mirbahai and Chipman, 2014).
Epigenetic changes in liver tissue from HCC patients following AFB1 exposure, have been demonstrated, e.g. gene-specific hyper-methylation (for example of RASSF1A, p16 (Zhang et al., 2006) and MGMT) as well as genome-wide hypo-methylation (Zhang et al., 2012). This appears frequently accompanied by a mutation in the p53 gene (Hsu et al., 1991, Hussain et al., 2007, Soini et al., 1996). In addition, specifically altered microRNA expression patterns (e.g. microRNA-221 and microRNA-122) have been observed in HCC patients (Fornari et al., 2008, Fornari et al., 2009, Gramantieri et al., 2008, Karakatsanis et al., 2013, Sun et al., 2013).
The underlying mechanisms by which AFB1 induces DNA methylation changes and microRNA expression changes important in chemical carcinogenesis, are complex, however some potential mechanisms have been proposed. Altered microRNA expression changes occur in response to DNA damage induced by a range of environmental stressors (Izzotti and Pulliero, 2014). Another potential mechanisms by which DNA methylation changes occur, is suggested to involve the preferential binding AFB1 to methylated CpG regions within the DNA, thereby inducing damage leading to conformational changes which may have an impact on the degree of methylation (e.g. causing genome-wide hypomethylation) (Zhang et al., 2003). Hyper- and hypo-methylation in especially the promotor region may impact on gene expression, while specifically hypo-methylation may also affect genome stability. In addition, DNA damage may be generated by AFB1-induced reactive oxygen species production, which may reduce the binding affinity of methyl-CpG binding protein 2 (MECP2), thus resulting in epigenetic changes (Valinluck et al., 2004). The MeCP2 protein binds to forms of DNA that have been methylated. The MeCP2 protein then interacts with other proteins to form a complex that turns off the gene. Once bound, MeCP2 will condense the chromatin structure, form a complex with histone deacetylases (HDAC), or block transcription factors directly (Yasui et al., 2007). The exact mechanisms by which AFB1-induced DNA methylation alterations and microRNA expression changes occur, however still remain complex and therefore need to be investigated in more detail. Furthermore, the effects of AFB1 exposure on whole genome DNA methylation and microRNA expression is not clear and therefore needs to be further explored.
However, neither in experimental studies on AFB1 carcinogenicity, nor in HCC patient studies in relation to AFB1 exposure, the persistence of epigenomic effects (including microRNA expression changes) of AFB1 exposure after removal of the exposure has yet been investigated. In addition, no attempts were made to integrate such a persistent epigenetic “footprint” with the transcriptome.
Consequently, in this present study, we hypothesize that by investigating the persistence of the AFB1-related epigenetic “footprint” together with perturbations of the transcriptome, we will be able to identify novel methyl DNA-microRNA-mRNA-interaction networks which are associated with early AFB1-induced onset of HCC. Simulation of chronic AFB1 exposure, leading to HCC, was initiated by repeated daily dosing. This is crucial since the half-life of AFB1 is 30 min and that of AFB1-DNA adducts in animals is less than 24 h (Lai et al., 2014, Qian et al., 2013). For that reason, primary human hepatocytes (PHH) were exposed to 0.3 μM of AFB1 for 5 days. Persistence of epigenetic effects was evaluated after 3 days of withdrawal of the carcinogen exposure. A 3-day wash-out period will permit the full removal of AFB1 and AFB1-DNA adducts from the PHH, in order to prevent effects taking place due to the presence of the carcinogen or its respective DNA adducts. Whole genome DNA methylation changes were measured by means of NimbleGen 2.1 deluxe promoter arrays, microRNA expression changes were measured through Agilent microRNA microarrays while whole genome transcriptomic analysis was performed using Affymetrix whole genome gene expression microarrays. Persistent hyper- or hypo-methylated genes with a concomitant alteration in their gene expression were investigated in more detail for their biological role within AFB1-induced onset of HCC using ConsensusPathDB (Kamburov et al., 2009) and when of relevance, by GeneCards (Rebhan et al., 1997).
Section snippets
Cell culturing, dose determination and aflatoxin B1 exposure
Cryopreserved primary human hepatocytes (PHH) were purchased from Life Technologies. Cells were cultured in pre-coated 24-well plates (700,000 cells/ml) in a 2-layer collagen sandwich (A11428-02, Gibco), according to the supplier’s protocol (Invitrogen). The following culture media were used: Hepatocyte Thawing Medium (HTM) for thawing (CM7500, Gibco), Williams’ Medium E (1x, no phenol red) (A1217601, Gibco) + Cell Maintenance supplement B kit (CM4000, Gibco) for plating and incubation. After
Determination of differentially methylated genes (DMGs)
After a pool of PHH was repetitively exposed for 5 days to 0.3 μM of AFB1 5639 genes were found to be differentially methylated (1896 genes were hyper-methylated and 3743 genes were hypo-methylated) (see part A and B of Supplementary-Table-3). After 5 days of exposure, the carcinogen was removed from the medium, and after 3 days of wash out 8131 differentially methylated genes were identified (4397 genes were significantly hyper-methylated and 3734 genes were hypo-methylated) (see part C and D
Discussion
In this study, a pool of PHH was exposed to 0.3 μM of AFB1 for 5 repetitive days. In addition, a 3-day wash-out period was inserted which permitted the full removal of AFB1 and AFB1-DNA adducts from the PHH, in order to avoid effects occurring due to the presence of the carcinogen or its respective DNA adducts. We were able to identify persistent effects on the epigenome as well as on the transcriptome level. No persistent effects could be observed in the microRNAome. Approximately 20% (386 out
Conclusion
In conclusion, we have identified persistent effects of AFB1 exposure on the epigenome and the transcriptome. By performing an integrative analysis, we were able to identify 6 important gene candidates, demonstrating a specific AFB1-related “epigenetic foot-print” as indicated by their persistent expression and DNA methylation change after the carcinogen exposure was ended. These 6 genes play a role in several biological processes which are known to be affected by AFB1 exposure and altered in
Funding
This work was supported by the Netherlands Genomics Initiative (NGI), the Netherlands Organization for Scientific Research (NWO), the Netherlands Toxicogenomic Center (NTC) [grant number 050-06-510], and the FP7 project DETECTIVE [grant number 266838].
Conflict of interest
The authors declare that there are no conflicts of interest.
Ethical standards
The manuscript does not contain clinical studies or patient data.
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