The implications of DNA methylation for toxicology: toward toxicomethylomics, the toxicology of DNA methylation

Abstract

Identifying agents that have long-term deleterious impact on health but exhibit no immediate toxicity is of prime importance. It is well established that long-term toxicity of chemicals could be caused by their ability to generate changes in the DNA sequence through the process of mutagenesis. Several assays including the Ames test and its different modifications were developed to assess the mutagenic potential of chemicals (Ames, B. N., Durston, W. E., Yamasaki, E., and Lee, F. D. (1973a). Carcinogens are mutagens: a simple test system combining liver homogenates for activation and bacteria for detection. Proc. Natl. Acad. Sci. U.S.A. 70, 2281-2285; Ames, B. N., Lee, F. D., and Durston, W. E. (1973b). An improved bacterial test system for the detection and classification of mutagens and carcinogens. Proc. Natl. Acad. Sci. U.S.A. 70, 782-786). These tests have also been employed for assessing the carcinogenic potential of compounds. However, the DNA molecule contains within its chemical structure two layers of information. The DNA sequence that bears the ancestral genetic information and the pattern of distribution of covalently bound methyl groups on cytosines in DNA. DNA methylation patterns are generated by an innate program during gestation but are attuned to the environment in utero and throughout life including physical and social exposures. DNA function and health could be stably altered by exposure to environmental agents without changing the sequence, just by changing the state of DNA methylation. Our current screening tests do not detect agents that have long-range impact on the phenotype without altering the genotype. The realization that long-range damage could be caused without changing the DNA sequence has important implications on the way we assess the safety of chemicals, drugs, and food and broadens the scope of definition of toxic agents.

FIG. 1.

Toxic agents affecting DNA methylation in dividing and nondividing cells, a model. In dividing cells, inhibition of DNMT1, the maintenance of DNMT, by putative toxic agent X during DNA replication will result in loss of methylation (CH₃) from certain sites. If the agent X is present in the next round of replication, both strands of DNA are demethylated at this position and a gene that was silenced by the methylated regulatory element is activated (indicated by the horizontal arrow). Once the site of methylation is lost on both strands, the situation is maintained for further cell divisions in the absence of the toxic agent X. The state of methylation acts as a memory in the genome to the transient exposure to substance X. The left side of the diagram shows the situation in a postmitotic cell. The balance of DNA methylation is defined by an equilibrium of methylating enzyme DNMT and demethylases. Putative toxic substance Y induces demethylase activity resulting in demethylation of a regulatory region and activation of transcription. Alternatively, substance X inhibits DNMT, tilting the balance between DNMTs and demethylases toward demethylase resulting in demethylation and activation of the gene (horizontal arrow). The new state of methylation is long-term maintained by the balance of DNMT and demethylases in the absence of the toxic agents.

FIG. 2.

The DNA methylation equilibrium; enzymatic targets of DNA methylation–modifying toxic agents. DNA is methylated by a transfer of a methyl moiety from the methyl donor SAM to the 5′ position on a cytosine ring by DNMT releasing S-adenosyl-homocysteine (SAH). SAM is regenerated by the following sequence of reactions: (1) hydrolysis of SAH to homocysteine by homocysteine hydrolase, (2) the methylation of homocysteine to methionine by methionine synthase, and (3) the adenylation of methionine to SAM by SAM synthetase. X-putative agents that could cause demethylation. Toxic agents (X) that inhibit either DNMTs or the four reactions involved in the synthesis of SAM will affect the DNA methylation equilibrium and reduce the drive toward DNA methylation, allowing an increase in DNA demethylation. Several demethylation reactions were suggested. Direct demethylation by a demethylase enzyme (dMTase) (MBD2 is a putative candidate) could release a methyl moiety (CH3) in the form of either methanol or formaldehyde. Alternatively, the methyl cytosine ring could be modified either by deamination catalyzed, e.g., by AID, or by the DNMT, which were shown to catalyze deamination of 5-methylcytidine in the absence of SAM or hydroxylation of the methyl moiety catalyzed by TET1. The modified base is then excised and repaired. Alternatively, the bond between the sugar and the base is cleaved (by glycosylases such as MBD4 or 5-methylcytosine glycosylase 5-MCDG) followed by repair. Repair proteins shown to be associated with demethylation were GADD45(a and b). Y-putative toxic agents that interfere with the different demethylation activities. These agents will reduce the drive toward demethylation and tilt the equilibrium to higher methylation activity resulting in hypermethylation.

FIG. 3.

The dynamic relationship between DNA methylation and chromatin structure, targets for toxic agents that alter DNA methylation patterns. The DNA methylation and chromatin modification equilibrium is laid down during embryogenesis. However, a balance of DNA methylation and demethylation activities as well as chromatin-activating modifications such as histone acetylation or inactivating modification such as histone deacetylation (catalyzed by HDACs) or H3K27 methylation (catalyzed by HMTase such as EZH2) dynamically maintains this pattern. The chromatin modification states and DNA methylation states are interrelated. Histone acetylation facilitates DNA demethylation, and histone H3K27 methylation facilitates DNA methylation. Therefore, agents that interact with the chromatin modification enzymes will also affect DNA methylation. Y-putative toxic agents that target DNMT1 or HDAC (such as TSA or valproic acid) will increase histone acetylation and facilitate DNA demethylation. X-putative toxic agents that target either the demethylase activities listed in Figure 2 or HAT will cause histone deacetylation and DNA hypermethylation.