In vitro hydroquinone–induced instauration of histone bivalent mark on human retroelements (LINE-1) in HL60 cells
Introduction
Benzene, a volatile aromatic hydrocarbon, is extensively used in industry even though it is recognized as a myelotoxin with leukaemogenic activity, representing a significant occupational risk (Khalade et al., 2010): it is also a well-known environmental pollutant, due to its release in the air through cigarette smoke and fumes of motor vehicles (Wallace, 1989). Exposure to high concentrations of benzene (> 100 ppm) leads to toxicity in the hematopoietic system, while acute myeloid leukaemia (AML) is the major oncogenic disorder associated with chronic exposure, although other forms of leukaemia have been reported (Aksoy, 1989, Atkinson, 2009, Khalade et al., 2010, Stenehjem et al., 2015). Metabolism of benzene plays a fundamental role in its toxicity (Meek and Klaunig, 2010, Zolghadr et al., 2012), and among the different metabolites, hydroquinone (HQ) is one of the most important (Snyder and Hedli, 1996), as it can induce several genetic and epigenetic changes (Liu et al., 2012). Aberrant patterns of DNA methylation, including loss of imprinting (LOI), gene-specific hyper- or hypo-methylation and global hypo-methylation are all common in AML and other tumour types (Lübbert et al., 1992, Bollati et al., 2007, Brait and Sidransky, 2011, Hamilton, 2011, Liu et al., 2011), and are important for transcriptional repression or activation of cancer-associated genes (Chen et al., 2004, Wilson et al., 2007). Nevertheless, the actual mechanism behind the influence of these aberrations on tumourigenesis remains unclear.
DNA methylation of repetitive elements is widely used in research and clinic as an indicator of global genomic methylation level, thanks to their high number of copies interspersed in the genome (Yang et al., 2004, Sahnane et al., 2015): the best-studied families are the long (LINE-1 or L1) and the short (SINEs, in particular Alu) interspersed nuclear elements (Byun et al., 2013). In human studies, differences in DNA methylation of L1 and Alu have been consistently demonstrated in response to a wide range of environmental exposures, including airborne pollutants (Bollati et al., 2007, Baccarelli et al., 2009, Peluso et al., 2012, Seow et al., 2012). LINE-1 DNA hypo-methylation was demonstrated in benzene-exposed healthy workers and partially in vitro in HQ treated cells (Bollati et al., 2007, Ji et al., 2010, Liu et al., 2012). Most of the studies conducted so far have been centred on DNA methylation, whereas only a few investigations have analysed the effects of environmental chemicals on histone modifications: only recently the relation with benzene exposure was explored (Baccarelli and Bollati, 2009, Philbrook and Winn, 2015).
DNA methylation is catalysed by three DNA methyl-transferases (DNMTs) (Portela and Esteller, 2010): aberrant expression levels of DNMTs induced by environmental carcinogens, accompanied by changes in global methylation, have been already observed during leukaemogenesis (Benbrahim-Tallaa et al., 2007, Ji et al., 2008, Liu et al., 2011). DNA methylation has a role in directing histone methylation, and vice-versa, with these two marks supporting each other to establish the chromatin environment (Greer et al., 2014, Rose and Klose, 2014): the bridge role between the two epigenetic levels is prevalently played by UHRF1 (ubiquitin-like with PHD and ring finger domains 1) (Bronner et al., 2013).
Histone methylation occurs on all basic residues (Fischle et al., 2008) and can happen at multiple different residues on the same histone catalysed by several histone methyl-transferases (Greer et al., 2014). Some histone marks are mutually exclusive: one of the most studied combined methylation is the distinctive signature that associates the repressive H3K27 tri-methylation (me3) mark and the activating H3K4me3 mark. This bivalent condition, harboured by many promoters in embryonic stem cells and essential during development, was first proposed by Bernstein et al. (2006) as a mechanism to silence developmental genes while keeping them poised for activation, and is lost after differentiation. Nowadays it is known to be present also in cancer, where can lead both to repression of previously active genes (following loss of H3K4me3), or activation of previously repressed sequences/genes associated with cancer progression (following loss of H3K27me3) (Chapman-Rothe et al., 2013, Voigt et al., 2013, Hahn et al., 2014).
The present work aims at addressing the epigenetic changes in response to sub-cytotoxic concentrations of HQ, by focusing prevalently on the possible alterations on chromatin and DNA methylation. Actually, in vitro demonstration of HQ activity on the epigenetic machinery is difficult to achieve due to the nature of the exposure: while acute effects (i.e. apoptosis activation and ROS production) are easily evaluable through single high-dose administrations of the agents (Zolghadr et al., 2012), and are thus well-studied, long-term exposures at low doses are more difficult to reproduce in laboratory. In this study, by setting up a long-term treatment with low-doses of hydroquinone within the levels of total HQ found in peripheral blood of benzene-exposed workers, we explore in vitro the differences between short and long-term exposure, to evaluate if long-term treatment with low-doses of hydroquinone might be sufficient to alter the epigenetic signature, thereby describing a poorly explored step in the mechanism of toxicity associated with benzene exposure.
Section snippets
Cell line and compounds
The MPO-positive AML cell line, HL60, was obtained from the American Type Culture Collection (ATCC, Rockville, MD, USA) and routinely cultured in suspension at 37 °C and 5% CO2 in RPMI medium supplemented with 10% foetal bovine serum (Euroclone, MI, IT) and 1% penicillin/streptomycin (Sigma-Aldrich, St. Louis, MO, USA). Hydroquinone (HQ, ≥ 99%) was purchased from Sigma-Aldrich, stocked at RT and dissolved in complete medium immediately before each treatment. Decitabine (DAC, Sigma-Aldrich) served
Results
Since the aim of our study was to investigate processes that take place in bone marrow, both catechol and hydroquinone were good candidates: however, we chose HL60 cell line as our model, which is MPO positive and can thus potentially metabolize HQ to benzoquinone, to possibly analyse the combined effects of both compounds. Exploring in vitro the differences between short and long-term exposure to HQ, in the effort to investigate if low-doses might be sufficient to alter the epigenetic
Discussion
In the last few years, several investigations have examined the relationship between exposure to environmental chemicals and epigenetics, and have identified numerous agents able to modify epigenetic marks. Most of the studies conducted so far have been centred on DNA methylation, whereas only recently a few have focused on the effects on histone modifications (Bollati et al., 2007, Wilson et al., 2007, Baccarelli and Bollati, 2009). Benzene and its metabolites are among the most studied, due
Conclusions
Exploring the epigenetic events occurring in chromatin, after in vitro treatment with concentrations of HQ representative of the levels produced by occupational exposures to airborne benzene, not able to alter cellular growth, we found the transient instauration in LINE-1 sequences of the combined signature H3K27me/H3K4me3, indicating the instauration of a poised chromatin conformation normally visible during development. The loss of these alterations after short-term treatments and the gradual
Conflict of interest disclosure
The authors declare no conflict of interest.
Transparency document
Acknowledgements
This work was supported by the Associazione Italiana per la Ricerca sul Cancro (AIRC), Investigator Grant 2012 (n. 13383). We thank Dr. Marzia B. Gariboldi for the helpful support with flow cytometry analysis.
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These authors contributed equally to this work.