**6. Epigenetics and environment exposure**

For many decades, it was assumed that chemicals are able to cause cancer only if they mutate the DNA. However, a growing body of scientific evidence reveals that this "carcinogenesis equals mutagenesis" paradigm is no longer accurate. Twenty years ago, Ashby and Tennant (1991) examined 301 chemicals tested by the US National Toxicology Program, and found that from 162 (53%) that were carcinogens, 64 (40%) were not genotoxic, illustrating the importance to focus on carcinogenic mechanisms other than genotoxicity. For many chemical agents, it has become increasingly clear that biological perturbations leading to neoplastic transformation may occur even in the absence of mutagenesis, via non-genotoxic, epigenetic changes. In addition, epigenetic changes may be relevant for tumor development and progression and ways that years ago seemed unimaginable. For example, Tanemura et al. (2009) revealed, for the first time, that CpG methylation in cutaneous melanoma is associated with tumor progression, and Nobeyama et al. (2007) demonstrated that tissue factor pathway inhibitor-2 (*TFPI-2*), a encoding a protein that suppresses the invasiveness of malignant melanoma, was methylated in 29% of metastatic lesions but in none of the primary tumors examined, pointing towards differences in gene expression and phenotypic characteristics between metastatic tumors and the primary tumor they originated from, a finding with significant therapeutic applications. The risk factors for sinonasal carcinoma include wood dust esposure, occupational exposure to chromium and nickel and its organic compounds (Luce & Leclerc, 2002)

Very high relative risks have been invariably found and 10-45 fold risks have been indicated for the sinonasal adenocarcinoma cell type in association to occupational exposure to hardwood dust, the risk related to softwood dust exposure is less clear. (Demers et al, 1997) Exposure to wood dust in the occupational setting is a common occurrence. It has been estimated that in the year 2000-2003 about 3.6 million workers were occupationally exposed to inhalable wood dust in the European Union and over half a million of these workers were estimated to be exposed to high levels (exceeding 5mg/m3) of wood dust (Kauppinen et al,, 2006). The terms "hardwood" and "softwood" refer to the taxonomic categorization of trees

changes in normal mucosa cells derived from surgical margins were detected in head and neck carcinomas (Martone et al, 2007). However, so far such changes have not been observed specifically in laryngeal cancers. In Paluszczak's paper (2011) evidence of a widespread occurrence of the aberrations in the profile of DNA methylation in laryngeal cancer patients is presented. Less than ten percent of cancer cases did not show any epigenetic changes in the normal mucosa samples. Gene methylation frequency was only slightly lower in the normal epithelium of epiglottis or trachea than in tumor cells. However, it should be taken into account that all the patients were exposed to similar laryngeal cancer risk factors. As discussed earlier, tobacco and alcohol are associated with the aberrations in the DNA methylation profile. The long-term exposure of patients to these factors could be responsible for the common appearance of epigenetic defects in a large field of upper respiratory tract mucosa. The existence of the epigenetically changed field in laryngeal cancers seems to be confirmed especially by such cases where lack of gene methylation in tumor cells was accompanied by the presence of hypermethylation in the normal epithelial cells although the percentage of patients with such gene methylation pattern was rather low.

For many decades, it was assumed that chemicals are able to cause cancer only if they mutate the DNA. However, a growing body of scientific evidence reveals that this "carcinogenesis equals mutagenesis" paradigm is no longer accurate. Twenty years ago, Ashby and Tennant (1991) examined 301 chemicals tested by the US National Toxicology Program, and found that from 162 (53%) that were carcinogens, 64 (40%) were not genotoxic, illustrating the importance to focus on carcinogenic mechanisms other than genotoxicity. For many chemical agents, it has become increasingly clear that biological perturbations leading to neoplastic transformation may occur even in the absence of mutagenesis, via non-genotoxic, epigenetic changes. In addition, epigenetic changes may be relevant for tumor development and progression and ways that years ago seemed unimaginable. For example, Tanemura et al. (2009) revealed, for the first time, that CpG methylation in cutaneous melanoma is associated with tumor progression, and Nobeyama et al. (2007) demonstrated that tissue factor pathway inhibitor-2 (*TFPI-2*), a encoding a protein that suppresses the invasiveness of malignant melanoma, was methylated in 29% of metastatic lesions but in none of the primary tumors examined, pointing towards differences in gene expression and phenotypic characteristics between metastatic tumors and the primary tumor they originated from, a finding with significant therapeutic applications. The risk factors for sinonasal carcinoma include wood dust esposure, occupational exposure to chromium and nickel and its organic compounds (Luce & Leclerc,

Very high relative risks have been invariably found and 10-45 fold risks have been indicated for the sinonasal adenocarcinoma cell type in association to occupational exposure to hardwood dust, the risk related to softwood dust exposure is less clear. (Demers et al, 1997) Exposure to wood dust in the occupational setting is a common occurrence. It has been estimated that in the year 2000-2003 about 3.6 million workers were occupationally exposed to inhalable wood dust in the European Union and over half a million of these workers were estimated to be exposed to high levels (exceeding 5mg/m3) of wood dust (Kauppinen et al,, 2006). The terms "hardwood" and "softwood" refer to the taxonomic categorization of trees

**6. Epigenetics and environment exposure** 

2002)

and not necessarly to the hardness of the wood. Woo dust is a complex mixture of compounds including a wide variety of biologically active substances, also genotoxic and carcinogenic compounds (Hanahan & Weinbrg, 2000). The particulate nature of the wooddust exposure plays a role in generating reactive species of oxygen (ROS) within cells and inducing DNA damage and evoking an inflammatory response (Bornholdt & Saber, 2007). Multiple mechanism of carcinogenesis have been proposes to be involved in the development of sinonasal cancer related to wood-dust exposure, but there is very little experimental or human data in the literature. The published findings have been based on a relatively limited number of cases, mostly involving adenocarcinomas. In these studies, high frequencies of DNA copy number chamges as detected by comparative genomic hybridization have been detected (Ariza et al,, 2004 and Korinth et al, 2005), while the mutation rates reported for the KRAS gene (Fratini et al, 2006 and Yom et al, 2005) and the p53 tumor suppressor gene have been lower (Perrone et al, 2003 and Licitra et al, 2004)). Initially, KRAS and HRAS mutations were found to be quite frequent in sinonasal cancer, with implications for histogenetic and prognostic significance (Yom et al, 2005), but recent studies show that tumors with KRAS mutation might represent only a small proportion of all sinanasal carcinomas (Bornholdt et al, 2008). Most of the studies having as subject p53 mutation have concentrated on the intestinal type of adenocarcinoma, and the numbers of cases studied have been rather low. In these studies, a variable occurrence of p53 mutations has been reported (18-60%) (Licitra et al, 2004 and Perrone et al, 2003). Some of the studies have also examined the accumulation of p53 in the cell nucleus in adenocarcinoma type of sinonasal carcer. The accumulation of p53 may reflect a p53 mutation.

With the exception of chromium, which forms DNA adducts, most carcinogenic metals are weak mutagens and act by epigenetic mechanisms (Arita & Costa 2009). Nickel, a metal linked to occupational and environmental exposures, has carcinogenic effects, despite the fact that it is not known to be a mutagen (Kasprzak et al, 2003; Chen et al, 2006; Ellen et al, 2009). *In vitro* and *in vivo* experiments reveal that nickel compounds silence gene expression by causing DNA methylation, an effect explained by the ability of nickel ions to substitute magnesium in the DNA phosphate backbone and to increase heterochromatin condensation (Arita & Costa 2009). The ability of Ni2+ ions to displace Mg2+ and cause chromatin condensation, establishing dense regions of heterochromatin that prevent accessibility to the respective genomic region, was also revealed by Ellen and colleagues (2009) with atomic force microscopy and circular dichroism spectropolarimetry. Chromatin condensation could in addition trigger DNA methylation in the compacted region, which also affects gene expression. When the silenced chromosomal region contains genes that are relevant to cancer initiation or progression, such as tumor suppressor genes or senescence genes, their inactivation can lead to disease (Ellen et al, 2009).

Nickel compounds also induce global changes in histone acetylation, methylation, and ubiquitylation. Kang et al. (2004) linked nickel to hypoacetylation to apoptosis for the first time, when they found hypoacetylation and demethylation of histones as potential mechanisms leading to apopotosis. Golebiowski and Kasprzak (2005) reported decreased acetylation of histones H2A, H2B, H3 and H4, in a time- and concentration-dependent manner, in human and rat cell lines exposed to nickel. Chen et al. (2006) revealed that nickel decreased a specific histone demethylase and by this mechanism, it increased global H3K9 mono- and dimethylation in several cell lines, and strongly suggested that this increased methylation causes the silencing of a transgene. This effect was dependent, *in vitro*, on iron

Epigenetics in Head and Neck Squamous Cell Carcinoma 189

In the first study that examined the effects of smoking on miRNA expression, Schembri et al. (2009) found 28 differentially expressed miRNAs in smokers, 82% of which were down regulated. One of them, mir-218, is also down regulated in several cancers, and the authors revealed that modulation of miR-218 levels lead to changes in the expression of its target genes. Xi et al. (2010) found that cigarette smoke condensate causes a significant and early increase in miR-31 that was apparent within 24 hours after exposure and persisted for 20

Epigenetic mechanisms regulate the interpretation of genetic information. As such, our knowledge of these mechanisms is essential for understanding the phenotypic plasticity of cells, both in the context of normal cellular differentiation and in human disease (Freinberg, 2007). Research over the past two decades has identified two major levels of epigenetic modification: DNA methylation and covalent histone modifications (Strahl & Allis, 2000 and Klose & Bird, 2006). DNA methylation is mediated by a family of enzymes termed DNA methyltransferases (DNMTs) (Goll & Bestor, 2005), while histone modification patterns are established and maintained by a diverse set of enzymes that add or subtract acetyl-, methyl-, and other modifications to various amino acids of histone proteins (Kouzarides, 2007). Both regulatory mechanisms cooperate to determine the expression potential of individual genes. For detection of cancel cells in body fluids, a high-sensitivity method is necessary. One way is mutation detection in cells because the exact location of a mutation within a gene is usually unknown, many primer sets are necessary for complete analysis. In contrast, aberrant methylation of DNA molecule of cancer cells, even in very few in number, can be sensitively detected by using Methylation-Specific PCR method (MSP), only with one set of PCR primer can be performed on chemically stable DNA, not on RNA (Herman et al,1996

Considering that some aberrant DNA methylation is present in early stages of carcinogenesis, there is a possibility that such demethylating agents may protect against some cancers (Laird et al, 1995). Demethylating agents are including DNMT1 inhibitors group (Azacitadine, Decitabine, Zebularine and MG98), procainamide, procain and EGCG (epigallocatechin-3-gallate) (Fang et al, 2003 and Villar-Garea et al, 2003). Inhibitors of DNMTs have been widely used in cell culture systems to reverse abnormal DNA hypermethylation and restore silenced gene expression. However, only limited success has been achieved in clinical trials with these drugs (Thibault et al, 1998 and Goffin & Eisenhouer, 2002). Also, nucleosides analog inhibitors of DNMTs may promote genomic instability and increase the risk of cancer in other tissues, because have many potential side effects such as myelotoxicity, mutagenesis and tumorigenesis (Jones &Taylor, 1980 and Gaudet et al, 2003). There is an attractive alternative for possible clinical use of non-

The use of these drugs raises questions regarding their potential to affect non-cancerous cells epigenetically. However, normal cells divide at a slower rate than malignant cells and incorporate less of these drugs into their DNA resulting in less of an effect on DNA methylation. Azacitadine and decitabine are labil and have acute hematological toxicities. Zebularine, a next generation DNA methylation inhibitor, might possibly overcome these

days after removal of the exposure.

and Laird, 2003).

nucleoside analog DNMT inhibitors.

**7. Challenges in epigenetic cancer therapy** 

and 2-oxoglutarate, and it is likely that it resulted from the nickel interfering with the iron moiety of the enzyme (Chen et al, 2006). In response to soluble nickel compounds at levels that had minimal cytotoxic effects, Ke et al. (2006) described three major histone modifications: H3K9 dimethylation, increased H2A and H2B ubiquitylation, and reduced histone acetylation, which was also associated with a transgene silencing.

An interesting example is provided by hexavalent chromium, to which half a million workers in the United States and several million individuals worldwide are occupationally exposed (Ali et al, 2011). Until recently, chromium was thought to cause cancer only through its ability to damage DNA (Arita & Costa 2009). Klein et al. (2002) reported for the first time that potassium chromate, a carcinogen, causes aberrant DNA methylation and silences a reported gene in a mammalian cell line. Ali et al. (2011) found higher rates of aberrant methylation in the promoters of three tumor-suppressor genes, APC (adenomatosis polyposis coli), MGMT (*O*6-methylguanine-DNA methyltransferase), and hMLH1 in lung cancers of chromate workers as compared to non-chromate lung cancer controls (95% versus 52%), with concordant methylation of multiple loci observed in more chromate lung cancers than in nonchromate ones (48% versus 12%). Chromate was also linked to post-translational histone modifications. Sun et al. (2009) found that hexavalent chromium at 5-10 μM concentrations causes global and local, gene-specific histone methylation changes in lung cancer, non-cancerous bronchial epithelial cells, and all respiratory tract which could impact tumorigenesis and tumor progression.

Benzene and aromatic hydrocarbons have increasingly emerged over the past few decades as environmental hazards. Exposure to benzene can occur in occupational settings, and also non-occupationally from coal or gasoline combustion products and cigarette smoke, which also contain polycyclic aromatic hydrocarbons. In the first study to show that low levels of a common environmental carcinogen are linked to epigenetic changes that occur in malignant human tumors, Bollati et al. (2007) reported that benzene, at low-level airborne exposures that are common in Western countries, causes epigenetic changes that reproduce modifications observed in human cancers. The authors examined 155 traffic police officers and gas station attendants, and 58 unexposed control subjects from Milan, Italy. The study revealed a dose-dependent global hypomethylation in the long interspersed nuclear element-1 (*LINE-1*) and *Alu*I repetitive sequences, in addition to hypermethylation at specific promoters, such as p15, which is also hypermethylated in patients with acute myeloid leukemia, in addition to hypomethylation in *MAGE-1*, a gene that is hypomethylated in many malignant tumors.

As a group of chemicals, polycyclic aromatic hydrocarbons (PAHs) include thousands of compounds ubiquitously distributed in the environment. An interesting fact about PAHs is that for a long time, the focus has been on their ability to cause genotoxic damage while their potential to induce epigenetic modifications was largely ignored (Upham et al, 1998). Benzopyrene, a prototype PAH, is a classic carcinogen found in vehicle emissions and in cigarette smoke - the mainstream smoke of a filter cigarette contains approximately 10 ng benzpyrene (Sherer et al, 2000), and exposure can also occur occupationally. In the first study to show that benzpyerene causes epigenetic changes, Sadikovic et al. (2008) conducted a genome-wide analysis after exposure and found 775 genes that were hypoacetylated and 1456 that were hyperacetylated. These modifications occurred in genes important for fundamental cellular processes, such as DNA replication, repair, and carcinogenesis.

and 2-oxoglutarate, and it is likely that it resulted from the nickel interfering with the iron moiety of the enzyme (Chen et al, 2006). In response to soluble nickel compounds at levels that had minimal cytotoxic effects, Ke et al. (2006) described three major histone modifications: H3K9 dimethylation, increased H2A and H2B ubiquitylation, and reduced

An interesting example is provided by hexavalent chromium, to which half a million workers in the United States and several million individuals worldwide are occupationally exposed (Ali et al, 2011). Until recently, chromium was thought to cause cancer only through its ability to damage DNA (Arita & Costa 2009). Klein et al. (2002) reported for the first time that potassium chromate, a carcinogen, causes aberrant DNA methylation and silences a reported gene in a mammalian cell line. Ali et al. (2011) found higher rates of aberrant methylation in the promoters of three tumor-suppressor genes, APC (adenomatosis polyposis coli), MGMT (*O*6-methylguanine-DNA methyltransferase), and hMLH1 in lung cancers of chromate workers as compared to non-chromate lung cancer controls (95% versus 52%), with concordant methylation of multiple loci observed in more chromate lung cancers than in nonchromate ones (48% versus 12%). Chromate was also linked to post-translational histone modifications. Sun et al. (2009) found that hexavalent chromium at 5-10 μM concentrations causes global and local, gene-specific histone methylation changes in lung cancer, non-cancerous bronchial epithelial cells, and all respiratory tract which could impact

Benzene and aromatic hydrocarbons have increasingly emerged over the past few decades as environmental hazards. Exposure to benzene can occur in occupational settings, and also non-occupationally from coal or gasoline combustion products and cigarette smoke, which also contain polycyclic aromatic hydrocarbons. In the first study to show that low levels of a common environmental carcinogen are linked to epigenetic changes that occur in malignant human tumors, Bollati et al. (2007) reported that benzene, at low-level airborne exposures that are common in Western countries, causes epigenetic changes that reproduce modifications observed in human cancers. The authors examined 155 traffic police officers and gas station attendants, and 58 unexposed control subjects from Milan, Italy. The study revealed a dose-dependent global hypomethylation in the long interspersed nuclear element-1 (*LINE-1*) and *Alu*I repetitive sequences, in addition to hypermethylation at specific promoters, such as p15, which is also hypermethylated in patients with acute myeloid leukemia, in addition to hypomethylation in *MAGE-1*, a gene that is

As a group of chemicals, polycyclic aromatic hydrocarbons (PAHs) include thousands of compounds ubiquitously distributed in the environment. An interesting fact about PAHs is that for a long time, the focus has been on their ability to cause genotoxic damage while their potential to induce epigenetic modifications was largely ignored (Upham et al, 1998). Benzopyrene, a prototype PAH, is a classic carcinogen found in vehicle emissions and in cigarette smoke - the mainstream smoke of a filter cigarette contains approximately 10 ng benzpyrene (Sherer et al, 2000), and exposure can also occur occupationally. In the first study to show that benzpyerene causes epigenetic changes, Sadikovic et al. (2008) conducted a genome-wide analysis after exposure and found 775 genes that were hypoacetylated and 1456 that were hyperacetylated. These modifications occurred in genes important for

fundamental cellular processes, such as DNA replication, repair, and carcinogenesis.

histone acetylation, which was also associated with a transgene silencing.

tumorigenesis and tumor progression.

hypomethylated in many malignant tumors.

In the first study that examined the effects of smoking on miRNA expression, Schembri et al. (2009) found 28 differentially expressed miRNAs in smokers, 82% of which were down regulated. One of them, mir-218, is also down regulated in several cancers, and the authors revealed that modulation of miR-218 levels lead to changes in the expression of its target genes. Xi et al. (2010) found that cigarette smoke condensate causes a significant and early increase in miR-31 that was apparent within 24 hours after exposure and persisted for 20 days after removal of the exposure.
