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Glycine N-methyltransferase (GNMT) is a mediator in the methionine and folate cycles, and is responsible for the transfer of a methyl group from S-adenosylmethionine (SAM) to glycine forming S-adenosylhomocysteine (SAH) and sarcosine. All the known DNA methyltransferases use SAM as a methyl donor thus, GNMT is critically involved in regulation of DNA methylation. Altered GNMT activities have been associated with liver pathologies including hepatocellular carcinoma. The homotetramer form of GNMT is enzymatically active, but the homodimeric form has been suggested as the 4S PAH-binding protein which may mediate CYP1A expression. To further understand the role of GNMT in benzo(a)pyrene (BaP)-related toxicity, full length Fundulus heteroclitus GNMT cDNA was cloned from adult liver. The open reading frame (ORF) of GNMT is 888 base pairs long and encodes a deduced protein of 295 amino acids which has 74% identity with human GNMT. Expression of GNMT mRNA was determined by quantitative RT-PCR. In unfertilized, 2 day postfertilization (dpf), and 3 dpf embryos GNMT was constitutively higher than in 4, 7, 10 or 14 dpf embryos. Embryos were also exposed to waterborne BaP at 10 and 100 μg · L−1, and by 10 dpf the higher BaP dose caused increased expression of GNMT mRNA. These results suggest that PAH exposure may alter expression of an important physiological methylation mediator. Future work will be necessary to determine enzyme level effects of BaP exposure as well.
Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous marine and aquatic contaminants. BaP exposure specifically can cause both fish embryo toxicity and liver carcinogenesis. While many of BaP's effects appear to be aryl hydrocarbon (AhR)- and CYP1-mediated toxicities (Billiard et al., 2007), we hypothesized that glycine N-methyltransferase (GNMT; EC126.96.36.199) could also be playing a role in PAH toxicity. GNMT is responsible for the transfer of a methyl group from S-adenosylmethionine (SAM) to glycine forming S-adenosylhomocysteine (SAH) and sarcosine, preserving the ratio of SAM/SAH, which is a sensitive indicator of methylation status. Altered expression of GNMT or SAM homeostasis is related to liver disease including hepatocellular carcinoma (HCC) and global or chromosome specific DNA methylation changes (Martinez-Chantar et al., 2008;Rowling et al., 2002). The homotetramer form of GNMT is enzymatically active and has unique glycine and folate binding sites. The homodimeric form, however, has been suggested as the 4S PAH-binding protein which may translocate to the nucleus and mediate CYP1A induction by various PAHs independent of the AhR (Bhat & Bresnick, 1997). More recently GNMT's ability to bind BaP was suggested as a protective mechanism in that binding would sequester BaP, and in turn, decrease adduct formation, CYP1A1 and GNMT activity and cytotoxicity (Chen et al., 2004). Previous studies in our laboratory have found that exposures to high concentrations of PAHs are capable of causing embryo toxicity and producing HCC in adult Fundulus heteroclitus. In this study, BaP was used to study effects on GNMT mRNA expression in Fundulus during embryo development to determine whether altered GNMT expression could be a potential mechanism involved in BaP-associated developmental toxicities.
Fundulus GNMT cDNA was cloned from adult liver mRNA. A partial Fundulus GNMT (764 bp) EST was found in the Fundulus EST database (FunnyBase, http://genomics.rsmas.miami.edu/funnybase/super_craw4/). Primers were designed based on this partial sequence. Both 5’ and 3’ RACE were used to amplify the respective ends of the cDNA, and then full length PCR products were generated using primers: forward: 5’-ACTAGCGGCAGCATGTC-3’, reverse: 5’-CCCGGATCTCAGATAAATCAG-3’. The full length GNMT cDNA contained a 5’ UTR of 63-base pairs (bp) long, and an open reading frame (ORF, Genbank Accession #FJ607947) of 888 bp. Because of sequence differences in the 3’ UTR between clones, genomic DNA was used as template to amplify the 3’ UTR of GNMT by using forward: 5’-CATGGAGCACACCGTCTAC-3’, reverse: 5’-CCCGGATCTCAGATAAATCAG-3’ primers. Three copies of the 3’ UTR with length of 794, 797 or 804 bp were cloned (data provided as a supplementary figure). Fundulus GNMT 3’ UTR was much longer compared to human (198 bp, #NM_018960) and zebrafish UTRs (351 bp, #NM_212816.1) and, to date, only Fundulus appears to have multiple copies (at least three) of GNMT 3’UTR. The significance of the variation in this region and between species is unknown.
Fundulus GNMT cDNA encodes a deduced protein of 295 amino acids with 74% and 79% identities with human (#Q14749) and zebrafish GNMTs (#AAH62527), respectively. Six single nucleotide polymorphisms (SNP) were discovered in the ORF region from two fish livers (represented by 13 and 9 colonies sequenced, respectively). SNPs in ORF positions 78 (C/T), 273 (A/G), 300 (A/C) did not change the amino acid translation, but SNPs in position 455 (A/T), 587 (A/G) and 841 (A/G) caused amino acid changes (Figure 1). However, as indicated in Figure 1, these amino acid changes were neither located in glycine nor SAM binding sites.
Fertilized eggs from Fundulus (caught in an uncontaminated site in the Newport River near Beaufort Inlet, NC, USA) were collected and randomly divided by 4.5 hpf into three waterborne treatment groups, namely control (dimethylsulfoxide, DMSO, 1 μl μ mL−1), 10 and 100 μg μ L−1 BaP. Ten embryos per pool (n = 3 – 6 pools) per time point were collected at 2, 3, 4, 7, 10, and 14 dpf. After 10 days of exposure, some embryos were returned to clean water prior to collection on day 14. RNA was isolated using Trizol reagent. Abundance of GNMT and 18S rRNA (internal control, #AF021880) transcripts was determined by quantitative real time RT-PCR with SYBR®Green on an ABI7500 using the 2−ΔΔCT method (ABI PRISM 7700 Sequence Detection System User's Manual). Statistical differences between treatments or times were determined on the linearized 2−ΔCT values. Real time primers for GNMT and 18S rRNA were: GNMT forward: 5’-CCTGCCGGACTTCAAAGGA-3’, reverse: 5’-TACGGTGGTCGATGATGAGAAT-3’; 18S forward: 5’-TGGTTAATTCCGATAACGAACGA-3’, reverse: 5’-CGCCACTTGTCCCTCTAAGAA-3’. Amplification efficiencies of the two primer pairs were not statistically different. In addition to melt curve analyses, the GNMT real time PCR product was confirmed by sequencing.
Unfertilized eggs had the highest expression of GNMT mRNA, suggesting maternal deposition into the egg and a possible important role of GNMT in early development. Constitutive GNMT mRNA expression decreased on 2 and 3 dpf but was still significantly higher than when expression leveled off between 4 and 14 dpf (Figure 2). BaP significantly induced GNMT mRNA expression over controls at 3, 10, and 14 dpf, but not at 2, 4 and 7 dpf (Figure 3). The GNMT induction was sustained even when 10 day BaP-exposed embryos were returned to clean water for another 4 days.
These results confirm that GNMT mRNA is present and inducible by BaP exposure in developing Fundulus. Therefore, GNMT may not only be important in fish developmental physiology, but also may represent a novel mechanism involved in PAH toxicity. Further work will be necessary to determine BaP effects on GNMT protein expression and enzyme activity and whether these changes, in turn, can impact DNA methylation status.
This research is supported by NIEHS funding: R01ES012710. We also want to thank Dr. Zhiqiang Pan from USDA Natural Products Utilization Research Unit, for his helpful suggestions on cloning and generous gifts of some cloning reagents. Parental Fundulus were kindly provided by Dr. Patricia McClellan-Green, NC State University.
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