Department of Pharmacology and Toxicology, B-440 Life Science Building, Michigan State University, East Lansing, Michigan 48824
Received December 3, 2001; accepted January 8, 2002
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ABSTRACT |
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Introduction |
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Epigenetics
Gene expression is not determined solely by DNA base sequence; this also depends upon epigenetic phenomena, defined as gene-regulating activities that do not involve a change to the base sequence (i.e., base-pairing is not altered) and can persist through one or more generations (Pennisi, 2001). Therefore, inheritance should be considered on a dual level. We should make a distinction between the transmission of genes from one generation to the next and in the somatic sense, from the mechanisms involved, in the transmission of alternative states of gene activity. Epigenetics describes the latter. One may view this as the study of mechanisms responsible for the temporal and spatial control of gene activity. Examples include changes in gene expression during development, segregation of gene activities such that daughters of an individual cell have different patterns of gene expression, and mechanisms to permit the somatic inheritance of a specific set of active and quiescent genes (Holliday, 1990
). Basically, gene expression can be regulated at multiple levels, i.e., DNA structure, gene transcription, and/or post-transcriptional modifications. Furthermore, epigenetic inheritance could function to shift patterns of gene expression to buffer an evolving biological system against changes in the environment. In this sense, adaptive epigenetic inheritance challenges the "central dogma" that information is unidirectional from DNA to protein and the idea that Darwinian random mutation and selection are the sole mechanisms of evolution (Monk, 1995
).
Four genetic mechanisms (DNA point mutation, deletion, rearrangement, and amplification) and two epigenetic mechanisms (DNA methylation, i.e., 5-methylcytosine content of DNA, and the preservation of DNA protein complexes) appear to account for the majority of heritable alterations of gene expression (Gruenert and Cozens, 1991). DNA methylation is also involved in imprinting, the phenomenon by which homologous genes are expressed differently according to whether they are inherited from the egg or sperm (Li et al., 1993
; Monk, 1995
). In contrast to classical Mendelian genetic theory, which states that there is equal inheritance of parental traits and predictable segregation of genetic characteristics among the progeny, genomic imprinting implies the differential expression of genes from the mother and the father (Monk, 1995
). The fact that 5-methylcytosine, like cytosine, base-pairs with guanine means that altered DNA methylation is not a mutation. In the current review, DNA methylation is the epigenetic mechanism that we will focus our attention upon.
DNA Methylation and the Regulation of Transcription
Approximately 4% of cytosine residues in mammalian DNA are methylated (Ehrlich et al., 1982). The predominant role of DNA methylation appears to be involved with transcriptional regulation leading to control over aspects of development, tissue-specific gene expression, expression of imprinted genes, and silencing of transposable elements (Rakyan et al., 2001
). In general, there is an inverse relationship between methylation and the amount of transcription; typically methylation in the promoter region of a gene leads to its transcriptional repression (Jones, 1999
). However, there are also cases in which nonpromoter hypermethylation, namely at differentially methylated portions of imprint control regions, is associated with an increase in gene expression, and vice versa (Thorvaldsen and Bartolomei, 2000
). For example, methylation of a particular region of the paternal allele of the imprinted gene Igf2 (insulin-like growth factor 2) blocks the binding of the CTCF protein, which would otherwise inhibit access of an enhancer protein to the Igf2 promoter region (Bell and Felsenfeld, 2000
). Thus, maintenance of distinct patterns of DNA methylation at promoter and non-promoter regions is essential for the regulation of normal mammalian gene expression. In addition, methylation is known to silence transcription of genomic "parasites," which if unchecked, could lead to decreased stability of the genome due to transcriptional interference, rearrangements within chromosomes, and/or translocations between chromosomes (Robertson and Wolffe, 2000
).
Methylation of promoter regions leads to inhibition of the binding of transcription factors directly and/or recruitment of the binding of proteins that act to inhibit the binding of the transcription factors to their cognate cis elements (Jones et al., 1998). In addition, methylated promoter regions are often coupled with regional histone deacetylation, suggesting that these two mechanisms may act in concert to accomplish transcriptional silencing (Ng and Bird, 1999
). Accordingly, studies have shown that certain methyl-CpG-binding proteins, such as MeCP2, bind specifically at methylated regions of DNA (Jones et al., 1998
). The Sin3 protein, which is bound to a deacetylase complex, can then bind the DNA-bound MeCP2, leading to histone deacetylation of the methylated regions of DNA (Jones et al., 1998
; Nan et al., 1998
).
Basically, methylation may be regulated by 3 mechanisms: de novo methylation of unmethylated cytosines, maintenance of methylation after DNA replication, and loss of DNA methylation of methylated cytosines by an enzymatic demethylation process (Laird, 1997). De novo methylation is accomplished by DNA methyltransferases Dnmt3a and 3b, and maintenance methylation is performed by Dnmt1, which acts preferentially at hemimethylated DNA (Robertson et al., 1999
). Regulation of methylation status is complex, and additional, as yet undiscovered regulatory factors are likely to be involved. Availability of methyl-group sources from S-adenosyl methionine is a requirement for both types of methylation (Kim, 2000
). Thus, regulation of normal methylation patterns requires the coordinated interplay of the numerous factors described above. An alteration in one or more of these may result in aberrantly methylated cells that exhibit a modified phenotype and are capable of expanding in a clonal fashion, and this might arise from threshold-exhibiting events (Goodman and Watson, 2002
). Additionally, extreme changes in methylation could result in modifications of gene expression and might lead to cell death.
Altered DNA Methylation: Carcinogenicity and Toxicity
Altered DNA methylation has been shown to contribute to carcinogenesis as well as to certain neuronal and developmental disorders (Robertson and Wolffe, 2001). Indeed, epigenetic misregulation of genes may be an important factor underlying a number of complex diseases (Petronis, 2001). Therefore, we believe it is reasonable to speculate that altered DNA methylation is a mechanism involved in multiple types of chemical-induced toxicities.
Today, cancer is the disorder most commonly associated with aberrant methylation, and chemical-induced alterations in methylation may play a variety of roles in carcinogenesis (Counts and Goodman, 1995; Robertson and Jones, 2000
). Actually, changes in methylation could be a mechanism involved in both the initiation and promotion stages of carcinogenesis (Goodman and Watson, 2002
). Altered DNA methylation may lead to carcinogenesis in several ways that are not mutually exclusive, including: (1) hypomethylation of promoter regions leading to over-expression of oncogenes, (2) hypermethylation of promoter regions leading to suppression of tumor suppressors, (3) hypermethylation leading to an increased incidence of deamination of 5-methylcytosine to thymine, leading to C to T point mutations in tumor suppressor genes and/or proto-oncogenes (Laird, 1997
). A fourth role that DNA methylation, may play in carcinogenesis is in the alteration of imprinted gene regulation, a common observation in certain types of human cancers (Jirtle, 1999
). Hypomethylation has been shown to be responsible for an increase in expression of the protooncogene c-myc (Pereira et al., 2001
). Conversely, a variety of tumor suppressor genes can be downregulated due to promoter-region hypermethylation. These include p16, E-cadherin, Bcl-2, O6-methylguanine-DNA methyltransferase (Belinsky et al., 1998
; Esteller et al., 2000; Leung et al., 2001
; Nagatake et al., 1996
; Nass et al., 2000). Hypermethylation may also contribute to the high percentage of C to T transitions in the TP53 tumor suppressor (Laird, 1997
). Furthermore, the capacity to maintain normal methylation patterns appears to be related inversely to susceptibility to liver tumorigenesis (Counts et al., 1996
).
The link between mental disorders and methylation aberrations indicates that DNA methylation plays a vital role with regard to normal functioning of the central nervous system (Robertson and Wolffe, 2000). A further indication for an important role of methylation in the brain is the observation of high levels of neuronal methyltransferase (Goto et al., 1993
). One of the first mental disorders to be linked to errors in methylation was fragile-X syndrome, a predominantly male form of mental retardation. Patients with this disorder display an increase in methylation at the CpG island upstream of the FMR1 (fragile-X mental retardation) gene coupled with a decrease in FMR1 expression (Robertson and Wolffe, 2000
). An additional mental disorder linked to alterations in methylation is Rett syndrome, an X-linked disorder responsible for a predominantly female form of mental retardation. This appears to stem from a mutation in the gene that encodes the methylcytosine-binding protein MeCP2 (Nan et al., 1997
). Furthermore, Prader-Willi and Angelman syndromes, both characterized by severe mental deficits, are linked to alterations in the methylation patterns of a differentially methylated region within the SNRPN promoter/exon1 region on the paternal and maternal alleles, respectively (Shemer et al., 2000
).
Beckwith-Wiedemann syndrome (BWS) is an example of a developmental disorder due to alteration of methylation-regulated imprinting mechanisms. BWS is characterized by developmental growth disorders, which, in some cases, is accompanied by increased expression of Igf2 (Issa and Baylin, 1996). Igf2 is typically a paternally expressed gene, but loss of imprinting may be caused by abnormal patterns of methylation (Maher and Reik, 2000
). Furthermore, certain methyltransferases may be essential in embryonic development; a homozygous null mutation of de novo methyltransferase Dnmt1 results in embryonic lethality in mice (Bestor, 2000
).
Additionally, ICF (immunodeficiency, centromeric instability, and facial anomalies syndrome) is both a mental and developmental disorder linked to altered methylation. ICF is characterized by immunosuppression, mental retardation, and particular facial characteristics (Wijmenga et al., 1998). Patients exhibit mutations in DNMT3, a de novo methyltransferase gene, which lead to abnormal hypomethylation in constitutive and facultative (X-inactive chromosome) heterochromatin (Xu et al., 1999
).
A limited number of agents have already been shown to elicit developmental defects via alterations in methylation. The cytidine analogue 5-aza-2`-deoxycytidine (dAZA) causes hypomethylation of DNA and administration of this drug results in developmental arrest in the early chick embryo, after inducing developmental abnormalities (Zagris and Podimatas, 1994). Administration of dAZA to pregnant mice results in perturbation of embryonal DNA synthesis, low fetal weight, and death of rapidly proliferating cells (Rogers et al., 1994
). Similarly, phenobarbital's capacity to cause rodent liver tumorigenesis is related, in part, to its ability to affect methylation (Counts et al., 1996
), and the carcinogenic actions of arsenite, dichloroacetic acid, and trichloroacetic acid appear to be related to their ability to induce hypomethylation and upregulate the oncogene c-myc (Chen et al., 2001
).
The maintenance of normal methylation patterns, including the re-programming that occurs during differentiation, is a crucial factor for both normal development and maintenance of the differentiated state (Reik et al., 2001; Rideout, et al., 2001
). Furthermore, controlled alterations in methylation are likely to be key components of normal plasticity in gene expression. DNA methylation plays a role in regulating the immune response (Mostoslavsky and Bergman, 1997
) Methylation is believed to play a role in the modulation of HLA (histocompatability leukocyte antigen) class-I genes as part of the normal T cell-mediated immune response (Serrano et al., 2001
). Thus, alterations in DNA methylation may underlie a variety of toxic responses, including but not limited to carcinogenesis.
The discussion presented above serves to highlight the importance of considering altered methylation as a possible mechanism underlying a variety of chemical-induced toxicities. These can occur as a consequence of heritable changes in the phenotype of target cells. Furthermore, alterations in gene expression due to epigenetic-based mechanisms might lead to cell death and this, too, can be a basis for toxicity. For instance, administration of dAza has been linked to the activation of the p53 DNA damage-response pathway that leads to a subsequent increase in cytotoxicity (Karpf et al., 2001). Therefore, assessment of DNA methylation status should be considered for inclusion as an ancillary component of both in vitro and in vivo tests to evaluate the potential of a chemical to cause toxicity and to provide information required to define the conditions under which the compound may be employed in a safe manner to benefit people and their environment. However, disruption of methylation is not necessarily problematic. In this context, it should be noted that compounds that alter DNA methylation might have therapeutic potential in anticancer therapy (Szyf, 2000
).
Assessment of DNA Methylation Status as a Component of Safety Assessment
It would not be appropriate to consider an assessment of the methylation status of a specific gene(s) as a routine component of testing protocols at this time. However, an evaluation of the overall methylation status and, possibly, methylation of particular regions (e.g., GC-rich regions of DNA) is both desirable and feasible as an initial step. This could lead to more thorough determinations, if warranted. A particular advantage here is that studies in vitro could be followed-up by examining putative target organs in vivo. At the time animals are sacrificed during routine subchronic and/or chronic studies in vivo, tissue samples can be taken and stored frozen prior to analysis, e.g., after evaluation of serum chemistry and histopathology. In both in vitro and in vivo experiments one could ask if altered DNA methylation might serve as a biomarker of toxicity and, if so, the dose (time)-response relationship could be ascertained.
Three general methods can be used to measure DNA methylation and each requires approximately 1 µg of DNA: the SssI methylase assay, arbitrarily primed PCR, and bisulfite modification, followed by either sequencing or methylation-specific PCR. The SssI methylase assay allows for the analysis of global (average) levels of DNA methylation. This procedure involves measuring the transfer of methyl groups from [3H]-methyl-S-adenosyl methionine to the 5-position of unmethylated cytosines that are 5` to guanine (Balaghi and Wagner, 1993). The arbitrarily primed PCR procedure allows for the analysis of methylation at GC-rich regions through the use of primers that bind preferentially to GC-rich sequences throughout the genome (Gonzalgo et al, 1997
). Finally, the bisulfite modification procedure converts unmethylated cytosine bases to uracil bases leaving methylated cytosines unaltered (Clark et al., 1994
: Feil et al., 1994
). The methylation status of the DNA can then be determined through sequencing (i.e., unmethylated cytosines will show up as thymines, while methylated cytosines will appear as cytosines) or methylation-specific PCR using primers specific for methylated or unmethylated sequences (Herman et al., 1996
).
We wish to emphasize the fact that we are suggesting the decision to determine methylation status should be made on a case-by-case basis and not simply added to a list of routine test requirements. Furthermore, changes in methylation are potentially reversible, and some modifications likely occur normally. Therefore, a chemical-induced change in methylation should not, a priori, simply be equated with toxicity.
Knowledge of the potential effect of a chemical on methylation status could aid in discerning its mechanism of action, e.g., a nongenotoxic mechanism. In this context, it is instructive to understand that altered DNA methylation may be a secondary, threshold-exhibiting mechanism (Goodman and Watson, 2002). Additionally, an understanding of the potential effect of a chemical on methylation status could facilitate a rational approach to dose setting and the selection of appropriate doses for risk assessment; e.g., if toxicity occurs only at high doses that cause altered methylation in the target organ(s), these data could aid in providing the basis for placing a proper emphasis on lower doses that are realistic with regard to potential human exposure.
In summary, incorporation of an assessment of methylation status as a component of an overall safety assessment may enhance, and indeed streamline, the process by facilitating the ability to (1) detect potential toxicity early (e.g., during in vitro studies and/or subchronic evaluations in vivo), (2) select rational doses for testing, especially in vivo, rather than placing excessive reliance upon doses that are too high, (3) define the shape of the dose-response curve, and/or (4) select appropriate doses for species-to-species extrapolation. For example, human cells are more capable of maintaining normal methylation status then rodent cells (reviewed in Goodman and Watson, 2002).
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ACKNOWLEDGMENTS |
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NOTES |
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