FORMALDEHYDE EFFECT ON DNA METHYLATION AND DEMETHYLATION

FORMALDEHYDE EFFECT ON DNA METHYLATION AND DEMETHYLATION Y Zhu, L Wang, HJ He, X Yang * College of Life Science, Central China Normal University, Wuhan 430079, China ABSTRACT Genomic DNA methylation is one of the most important epigenetic mechanisms. Existing data of indoor air formaldehyde effect on DNA methylation patterns is limited. S-adenosylmethionine, the sole methyl donor in this reaction, is generally connected to the tetrahydrofolate and the methionine homocysteine cycle and one carbon metabolism. In mammalian systems, as a normal intermediary metabolite, endogenous formaldehyde remains in equilibrium in blood and tissues. There are two ways that endogenous FA can be involved in one carbon pool. One is the production of 5, 10-methlene-THF formed by FA and THF. The second way is through the formation of 10-formyl-THF. Experiments indicate that the oxidation of 10-formyl-THF to 10-formyl-dihydrofolate takes place and 10-formyl- dihydrofolate is subsequently converted to dihydrofolate. Therefore, in light of those circles, we hypothesize that endogenous FA can act as one carbon group to participate in the process of DNA methylation or may cause demethylation through free radical injures. INDEX TERMS Endogenous formaldehyde, DNA methylation, DNA demethylation INTRODUCTION Formaldehyde is regarded as an important indoor air chemical pollutant for its extensive sources, high level, long-term and high toxicity. Many studies showed that indoor air formaldehyde induces many harmful effects on human being, such as carcinogenicity. In most epidemiological studies, the potential association between exposure to formaldehyde and cancer of the respiratory tract has been examined. In case-control studies, while some-time no increase was observed overall(vaughan et al, 1986), significantly increased risks of nasopharyngeal cancer (up to 5.5-fold) were observed among workers with 10-25 years of exposure or in the highest exposure category in three out of four investigations (West et al.,1993). Nowadays, it ranks in AI (human carcinogen) in WHO document (2004). The harmful degree of FA on human health is determined by inner dose, which includes inhaled formaldehyde and endogenous formaldehyde. Therefore, in order to exactly explore the toxicity of formaldehyde on human tissue cells, we must not ignore the effect of endogenous formaldehyde. The fundamental effect of endogenous formaldehyde to tissue cells is in process of our laboratory, so that we can exactly realize the harmful effect of indoor air formaldehyde in the future. DNA methylation is the most common eukaryotic DNA modification and is one of the epigenetic (alteration in gene expression without a change in nucleotide sequence) phenomena. Genome stability, allele-specific expression of imprinted genes and masking of transposons and X chromosome inactivation are all clearly dependent of cytosine methylation. The effect of DNA methylation to oncogenesis appears to be important in current achievements. And endogenous formaldehyde which keeps quantity stable via one-carbon pool in blood and tissues is normal metabolic in our bodies (Heck HD, Casanova-Schmitz M, et al. 1985). In light of those circles, we hypothesize that endogenous FA can act as one carbon group and participate in the process of DNA methylation. On the other hand, endogenous FA exposure can indirectly induce DNA damage (directly by oxygen radical injury), followed by the occurrence of 8-hydroguanosine. If cells fail to withstand the damage or the repair mechanism is inefficient, this lesion can result in mutation in genome. Consequently DNA methylation pattern will be influenced. So, endogenous FA may cause demethylation through free radical injures. This hypothesis can be validated in two ways. Firstly, analyzed the global genome methylation by high performance liquid chromatography (HPLC); secondly, in parallel, using the method of MSP (methylation-specific PCR) to evaluated changes in expression of the tumor suppressor gene p16inka. DNA METHYLATION DNA methylation in eukaryotes involves addition of a methyl group to the carbon 5 position of the cytosine ring (Bird A. 1992). This reaction is catalyzed by DNA methyltransferase in the context of the sequence 5 -CG-3, which is also referred to as a CpG dinucleotide. In most vertebrates, 60-90% of the cytosines at CpG dinucleotides * Corresponding author email: yangxu@mail.ccnu.edu.cn, guyangxu@yahoo.com 3817

are methylated. During evolution, the dinucleotide CpG has been progressively eliminated from the genome of higher eukaryotes and is present at only 5% to 10% of its predicted frequency. Cytosine methylation appears to have played a major role in this process, because most CpG sites lost represent the conversion through deamination of methylcytosines to thymines. Approximately 70% to 80% of the remaining CpG sites contain methylated cytosines in most vertebrates, including humans. In contrast to the rest of the genome, smaller regions of DNA, called CpG islands, ranging from 0.5 to 5 kb and occurring on average every 100 kb, have distinctive properties. These regions are unmethylated, GC rich (60% to 70%), have a ratio of CpG to GpC of at least 0.6, and thus do not show any suppression of the frequency of the dinucleotide CpG. DNA methylation plays important roles in gene expression. It may suppress gene transcription by direct or (and) indirect inhibition of transcription factor binding (Singal R, Ginder GD 1999). The density of DNA methylation, the intensity of promoter and the relative location of methylated DNA and promoter can all influence this inhibitory effect. Decreased levels of overall genomic methylation are common findings in tumorigenesis. This decrease in global methylation appears to begin early and before the development of frank tumor formation. Apart from the overall genomic hypomethylation, specific oncogenes have been observed to be hypomethylated in human tumors. DNA DEMETHYLATION Two kinds of DNA demethylation can be distinguished: globe and site specific demethylation, which plays important roles in embryo development, ensuring the needed genes are expressed in specific cells (Singal R & Ginder GD. 1999). There are two potentially biochemical mechanisms allowing DNA demethylation (Kress C, et al 2001): passive demethylation and active demethylation. Passive demethylation is related to DNA semi conservative replication. When the newly synthesized DNA strand fails to be methylated, for example, in the absence of DNA methylatransferase, it turns into semi-methylated double strand. After a new round of replication, half of the DNA becomes demethylated, thus changing the existed methylation pattern. It has been described that cytosine analogue 5-azadeoxycytidine initiates this sort of demethylation by covalently trapping methylatransferase, and stimulated the expression of some proto-oncogenes. An active demethylation may be catalyzed by so-called demethylation transferase. Bhattachrya and his colleagues found that MBD2 can lead to demethylation; therefore they regarded MBD2 as demethyltransferase (Bhattacharya SK, Ramchandani S, et al. 1999). DNA METHYLATION WITH THF AND METHIONINE SYNTHESIS CYCLES Reduced from folic acid, tetrahydrofolate (THF) acts as one-carbon resources in a wide variety of living organisms. Mammals cannot produce THF but gain it from food or cleavage molecules in intestines. Aberrant DNA methylation has been considered as a leading mechanism by which folate deficiency enhances colorectal carcinogenesis (Jacob RA et al. 1998). THF can enzymatically convert into 5,10-dimethylene-THF, 5-methyl-THF and finally THF. The last step is catalyzed by coenzyme Vitamin B12 and regulated by methionine synthase. The methyl groups, generated from 5-methyl-THF to THF, participate into methionine cycle. Methionine is one of the three amino acids that contain sulfur element and a vital methyl donor in organisms. Animal experiments showed that after having food deficient of folic acid, methionine and Vitamin B12 for a week, the level of SAM in rat hepatic cells decreased significantly; the amount of mrna transcripted from some proto-oncogenes such as c-myc, c-fos increased and showed hypomethylation. However, the methylation patterns return to normal with the supplement of adequate folate (Wainfan E & Poirier IA. 1995). It should be noticed that SAM, but not methionine can donate methyl group directly. SAM is generated by reaction of adenosine and methionine. SAM is the sole methyl donor in the process of DNA methylation. Catalyzed by methyltransferase, SAM shifts one methyl group and converts into SAH and L-homocysteine subsequently. Homocysteine is methylated also through the active form of Vitamin B12, regenerating methionine. FORMALDEHYDE AND DNA METHYLATION Early researches indicated that formaldehyde, whether from external or produced in vivo by metabolic reaction, is in equilibrium in mammalian blood and organs, which means that endogenous formaldehyde is a normal metabolite and may be capable of some biological roles. Formaldehyde can be generated via microsomal cytochrome P-450 dependent oxidation. A wide variety of exogenous compounds having N-methyl, O-methyl, or S-methyl group are subjected to this oxidative 3818

demethylation. Another way of formaldehyde production is given by the action of SSAO (semicarbazide-sensitive amine oxidase) which have endogenous substrates such as aminoacctone and methylamine (Kalász H. 2003). There are three main metabolism pathway of formaldehyde: be oxidated to formate and finally CO2, imediates DNA-protein crosslinks; bind to 10-formyl-THF and become a source for one carbon pool (Conaway CC, Whysner J, et al. 1996). Formaldehyde may participate in DNA methylation mainly via one-carbon pool and tetrahydrofolate cycle. Two assumed pathways may be involved. The first one is that in the researches on serine hydroxymethyltransferase crystal structure, J. Neel Scarsdal found that formaldehyde, generated in the transformation of serine and glycin, could bind to folic acid and form 5,10-dimethylene-THF(Scarsdale JN et al. 2000). The second one is related to its incorporation reaction. The balanceable system of formaldehyde in blood suggested the possible buffer system. McGilvery held that this system was maintained by the revesible convertion of 10-formal-THF and THF(McGilvery RW. 1979). Formaldehyde binds to THF and then 10-formayl-THF forms. Part of 10-formayl-THF demethylated to THF supported by the energy from ATP hydrogenesis; the left part systematically transferred to 5, 10-formayl-THF, which act as one-carbon unit source (Baggott JE & Tamura T. 2001). To sum up, the process as shown in Fig.1. FORMALDEHYDE AND DNA DEMETHYLATION Enormous studies have indicated the toxicity of formaldehyde, such as genotoxic, carcinogenesis, cytotoxicity, immountoxicity, neurotoxicity and so on. The main aspects of cytotoxicity present as DNA oxidative injure, promotion of cell proliferation and toxic inflamitation. The key point is that formaldehyde indirectly mediates DNA oxidative injure through free radical species rather than directly cause DNA damage. Zhuge Xi, et al found that the reaction of formaldehyde with DNA in vitro is weak, but the oxidative ability is enhanced and the reaction could produce a number of 8-OHdG adducts mediated by the Fe 2 +. The animal experiment showed that formaldehyde could cause the oxidative DNA damage of rat lung tissues, which suggested that formaldehyde has the stronger genotoxicity (Zhuge Xi, et al. 2004). Cerda S et al (1997) found that replacement of guanine with the oxygen radical adduct and hydroxyguanine profoundly alters methylation of adjacent cytosines, suggesting a role for oxidative injurly in the formation of aberrant DNA methylation. When the cells are unable to withstand the damage or the repair mechanisms can not correct the lesion, the existed methylation patten alters without changing base sequence. This process can lead to the loss of methylation via passive demethylation. folateacid 10-formyl-dihydrofolate dihydrofolate 10-formyl-THF tetrahydrofolate methionine formaldehyde 5,10-methylene-THF 5-methyl-THF B 12 methyl S-adenosylmethionine S-adenosyl-L-homocysteine homocysteine methylation methyl Figure 1. The process that formaldehyde may bear a part in the course of DNA methylation via one-carbon pool. TECHNIQUES TO STUDY DNA METHYLATION Early techniques to study site-specific DNA methylation relied primarily on the inability of methylation-sensitive type II restriction enzymes to cleave sequences containing one or more methylated CpG sites, combined with Southern hybridization (Bird AP & Southern EM. 1978, Waalwijk C & Flavell RA. 1978, McGhee JD & Ginder GD. 1979). This method requires large amounts of high molecular weight DNA, detects methylation only if more than a few percent of alleles are methylated, and only provides information about those CpG sites found within the 3819

recognition sequence of methylation-sensitive restriction enzymes. Methylation-specific PCR (MSP) is a simple rapid and inexpensive method to determine the methylation status of CpG islands (Herman JG & Baylin SB 1998). This approach allows the determination of methylation patterns from very small samples of DNA, including those obtained from paraffin-embedded samples. MSP utilizes the sequence differences between methylated alleles and unmethylated alleles which occur after sodium bisulfite treatment. The frequency of CpG sites in CpG facilitates this sequence difference. Primers for a given locus are designed which distinguish methylated from unmethylated DNA in bisulfite-modified DNA. Since the distinction is part of the PCR amplification, extraordinary sensitivity, typically to the detection of 0.1% of alleles can be achieved, while maintaining specificity. Results are obtained immediately following PCR amplification and gel electrophoresis, without the need for further restriction or sequencing analysis. MSP also allows the analysis of very small samples, including paraffin-embedded and microdissected samples. The global genome methylation is analyzed by HPLC (Wainfan E et al. 1992). CONCLUTION Evidences show that the loss of DNA methylation equilibrium commonly exist in carcinogenesis. Formaldehyde is a human carcinogen, and how it leads to tumor is still unknown. Therefore, further researches will be required to understand how formaldehyde can lead to abnormalities of the methylation machinery and what components of this machinery will be appropriate targets for therapeutic intervention. ACKNOWLEDGEMENTS This work was supported by the Ministry of Science and Technology for China National Key Projects (2004BA704B0105, 2004BA809B0604, 2004BA809B0605). REFERENCES Baggott JE., Tamura T., 2001. Metabolism of 10-formaldihydrofolate in humans. Biomed Pharmacother, 55:454-7. Bhattacharya SK., Ramchandani S., Cervoni N., Szyf M., 1999. A mammalian protein with specific demethylase activity for mcpg DNA. Nature 397(6720): 579-583. Bird A., 1992. The essentials of DNA methylation. Cell,70; 461-467. Bird AP., Southern EM., 1978. Use of restriction enzymes to study eukaryotic DNA methylation: I. The methylation pattern in ribosomal DNAfrom Xenopus laevis. J Mol Biol,118:27. Cerda S., Weitzman SA., 1997. Influence of oxygen radical injury on DNA methylation Mutat Res,386(2): 141-52. Conaway CC., Whysner J., Verna LK., et al. 1996. Formaldehyde mechanistic Data and risk assessment: endogenous protection from DNA adduct formation. Pharmacol Ther, 71: 29-55. Heck Hd.,Casanova-Schmitz M., Dodd PB. 1985. Formaldehyde (CH2O) concentration in the blood of humans and Fischer 344 rats exposed to CH2O under controlled conditions. Am Ind Hyg Assoc J 46:1 3. Herman JG., Baylin SB. 1998. Methylation Specific PCR, in Current Protocols in Human Genetics,29-55. Jacob RA., Gretz DM., Taylor PC. et al. 1998. Moderate folate depletion increases plasma homocysteine and decreases lymphocyte DNA methylation in post- menopausal women. J Nut,128: 1204-1212. Kalász H. 2003. Biological Role of Formaldehyde, and Cycles Related to Methylation, Demethylation, and Formaldehyde Production. Medicinal Chemistry, 3:175-192. Kress C., Thomassin H. 2001. Local DNA de- methylation in vertebrates: how could it be performed andtargeted. FEBS Letters, 494:135-140. McGhee JD., Ginder GD. 1979. Specific DNA methylation sites in the vicinity of the chicken beta-globin genes. Nature, 280:419. McGilvery RW. 1979. Fuel for breathing. Am Rev Respirs. Di,119:85-88. Scarsdale JN, Radaev S, Kazanina G, et al, 2000. Crystal Structure at 2.4 Å Resolution of E. coli Serine Hydroxymethyl-transferase in Complex with Glycine Substrate and 5-Formyl Tetrahydrofolate. J Mol Biol, 296:155-168. Singal R., Ginder GD. 1999. DNA Methylation. Blood,93(12): 4059-4070. Vaughan TL., Strader C., Davis S. et al. 1986. Formaldehyde and cancers of th pharynx, sinus and nasal cavity: I. Occupational exposures. Int J Cancer 38:677-683. Waalwijk C., Flavell RA. 1978. MspI, an isoschizomer of hpaii which cleaves both unmethylated and methylated hpaii sites. Nucleic Acids Res, 5:3231. Wainfan E., Dizik M., Stender M. et al. 1992. Methyl group in carcinogenesis:effects on DNA methylation and gene expression. Cancer Res 52:2071-2077. 3820

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