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Vitamin Metabolism Laboratory, Jean Mayer U.S. Department of Agriculture Human Nutrition Research Center on Aging at Tufts University, Boston, MA 02111 and * Department of Pathology and Laboratory Medicine, University of North Carolina, Chapel Hill, NC 27599
4To whom correspondence should be addressed. E-mail: schoi{at}hnrc.tufts.edu.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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KEY WORDS: • cystathionine ß-synthase • DNA methylation • S-adenosylmethionine • S-adenosylhomocysteine • mice
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). In remethylation, homocysteine acquires a methyl group from N-5-methyltetrahydrofolate or from betaine to form methionine. The reaction with N-5-methyltetrahydrofolate occurs in all tissues and is vitamin B-12 dependent, whereas the reaction with betaine is confined mainly to the liver and is vitamin B-12 independent. In the transsulfuration pathway, homocysteine condenses with serine to form cystathionine in an irreversible reaction catalyzed by the pyridoxal-5'-phosphate (PLP)5
-containing enzyme, cystathionine-ß-synthase (CBS). Cystathionine is then hydrolyzed by a second PLP-containing enzyme, 
-cystathionase, to form cysteine and 
-ketobutyrate (2
). A decreased rate of metabolism through either of these pathways can lead to homocysteinemia and homocystinuria.
Carson and Neill (3
) first described homocysteinuria in mentally retarded individuals and Mudd et al. (4
) subsequently reported that the primary defect in homocysteinuria was a defect in the CBS enzyme. CBS deficiency is the most common inborn error of sulfur amino acid metabolism. Patients with this enzyme deficiency are characterized biochemically by severe hyperhomocysteinemia and homocysteinuria, and clinically by premature arteriosclerosis and thromboembolism (5
).
In 1995, Watanabe et al. (6
) reported the generation of a knockout mouse that was deficient in CBS. The rationale behind the creation of this mutant was the growing evidence that elevated concentrations of total homocysteine (tHcy), in blood are associated with an increased risk of vascular disease. The homozygous mutant mice did not exhibit histological signs of atherosclerosis, in spite of the fact that their homocysteine levels were significantly greater than those of the wild-type and heterozygous strains (6
). However, it was shown recently that heterozygous mice, which have slightly increased concentrations of plasma tHcy, have impaired endothelial function (7
,8
). Although this animal model does not show the expected arteriosclerosis, it provides a unique opportunity to study the effect of elevated tHcy on one-carbon metabolism.
S-Adenosylmethionine (SAM), S-adenosylhomocysteine (SAH), and the SAM/SAH ratio have been used to evaluate the "integrity" or "capacity" of biological methylation (9
–11
), including DNA methylation. In the present study, we evaluated the effect of elevated plasma tHcy induced by CBS deficiency on one-carbon metabolism by comparing SAM and SAH concentrations as well as DNA methylation status in different tissues of homozygous mutant and wild-type mice.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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This study was approved by the Institutional Animal Care and Use Committee of the Jean Mayer USDA Human Nutrition Research Center on Aging at Tufts University. CBS-deficient mice were obtained from the University of North Carolina at Chapel Hill, where these mice were developed. All mice were maintained in commercially available caging and bedding and consumed an ad libitum standard (or autoclaved) food (Teklad 7012, Madison, WI) and water. The tip end of the tail was clipped from each mouse and DNA was extracted to determine the genotype. For mice < 14 d old, the tail was clipped without anesthesia and an ear punch was performed for animal identification purposes. No tail clips were done on 14- to 21-d-old mice. For mice that were > 21 d old, the tail clip was done under anesthesia; at the same time, the ear was punched for identification purposes. To maintain the colony, there were four breeding generations per year with 2 males and 4 females bred per generation (24 mice/y).
Ten homozygous mutant and 10 age- and sex-matched wild-type mice were used in this study. The mice were between 3 and 9 mo old and the male to female gender ratio was 4 to 6. After the mice were anesthetized with carbon dioxide, blood was drawn by cardiac puncture, they were killed by exsanguination, and liver, kidney and brain were harvested.
CBS genotype determination.
DNA was extracted from the tail clip using a commercially available DNA extraction kit (Invitrogen-Easy DNA kit, San Diego, CA). Polymerase chain reaction (PCR) was carried out using the primers 5'-GAAGTGGAGCTATCAGAGCA-3'; 5'-TGGCTCTTGGTTCTGAAACC-3' and 5'-GAGGTCGACGGTATCGATA-3' to generate a 450-bp fragment in the homozygous mutant and an 800-bp fragment in the wild-type mice (6
). The PCR products were electrophoresed on a 1.5% agarose gel and visualized by ethidium bromide staining.
Measurement of homocysteine.
Total plasma homocysteine was determined by HPLC using fluorometric detection as described by Araki and Sako (12
).
Measurement of SAM and SAH.
SAM and SAH concentrations were measured by HPLC with UV detection using the method described by Fell et al. (13
) with modification (14
).
DNA methylation assay.
The methylation status of CpG sites in genomic DNA was determined by the in vitro methyl acceptance capacity of DNA using [3H-methyl]SAM as a methyl donor and a prokaryotic CpG DNA methyltransferase, as previously described (15
). Briefly, 2 µg of DNA was incubated for 2 h at 37°C with 185 kBq of [3H-methyl]SAM (New England Nuclear, Boston, MA), 4 U of Sss1 methylase (New England Biolabs, Beverly, MA), 1X Sss1 buffer (50 mmol/L NaCl, 10 mmol/L Tris-HCl, 10 mmol/L EDTA, 1 mmol/L dithiothreitol, pH 8.0) in a total volume of 50 µL methylation mixture. Sss1 methylase was denatured by heating at 65°C for 20 min. The incubation mixture was applied onto the discs of Whatman DE-81 ion exchange filters (Fisher Scientific, Springfield, NJ) using a vacuum filtration apparatus; the discs were then washed with 0.35 mol/L Na2HPO4 for 45 min. The discs were dried at 95°C for 30 min, and the resulting radioactivity of the DNA retained in the discs was measured by scintillation counting using a nonaqueous scintillation fluid. All analyses were done in triplicate and the three values were averaged.
Statistical analysis.
To determine the effect of CBS deficiency on different aspects of one-carbon metabolism, we compared plasma tHcy, SAM, SAH, the SAM/SAH ratio and the methylation status of homozygous mutant mice with those of wild-type mice using paired t tests. For some of the variables, a log transformation was applied before analysis when it improved the normality of the differences between the two groups. These variables are identified with an asterisk in Tables 1
and 2
. Values in the text are means ± SD.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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The plasma tHcy level of food-deprived homozygous mutant mice (271.1 ± 61.5 µmol/L) was greater than that of wild-type mice (7.4 ± 2.9 µmol/L) (P < 0.001).
SAM and SAH concentrations and the SAM/SAH ratio in different tissues.
SAM levels in homozygous mutant mice were greater in liver (P < 0.001) and tended to be greater in kidney (P = 0.07), but did not differ from wild-type mice in brain (P = 0.30) (Table 1)
. In contrast, SAH levels in homozygous mutant mice were higher in all three tissues compared with those of wild-type mice: 8 times higher in liver (P < 0.001), 8 times higher in kidney (P < 0.001) and 190 times higher in brain (P < 0.001). The SAM/SAH ratio in homozygous mutant mice was significantly lower in all three tissues compared with that of wild-type mice: 80% lower in liver (P < 0.001), 87% lower in kidney (P < 0.001) and essentially 100% lower in brain (P < 0.001).
DNA methylation status in different tissues.
The DNA methyl acceptance capacity in homozygous mutant mice was higher in liver DNA (P = 0.037), tended to be higher in kidney DNA (P = 0.077) and did not differ in brain (P = 0.46) compared with mean DNA methyl acceptance capacity in wild-type mice (Table 2)
.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). To investigate the effect of severe hyperhomocysteinemia, we chose homozygous mutant mice instead of heterozygous mice, which have a mild elevation of plasma tHcy concentration (6
,16
). In the present study, homozygous mutant mice lacking the CBS enzyme had a 35-fold higher plasma tHcy concentration than wild-type mice. These results agree with previous studies (6
), and suggest that this mouse model is a good model for severe hyperhomocysteinemia resulting from CBS deficiency.
In vertebrate genomes, 
4% of cytosine residues are modified postsynthetically to 5-methylcytosine. The cell strictly maintains its particular patterns of methylated residues, and DNA methylation is thought to play a role in the regulation of gene expression and gene integrity (17
). There is considerable evidence suggesting that aberrant DNA methylation plays an integral role in oncogenesis. SAM, a universal methyl donor, reflects DNA methylation status because low levels of SAM impair many methylation reactions (18
,19
). Homocysteine, SAH and the SAM/SAH ratio have also been considered to be indicators of DNA methylation status (9
,10
). However, results have been inconsistent in many animal studies. In rodent models of hepatocarcinogenesis, diets deficient in sources of methyl group donors (choline, folate, methionine and vitamin B-12) decreased hepatic SAM levels and subsequently induced hepatic DNA hypomethylation (19
,20
). However, in the Sprague-Dawley rat model of moderate folate deficiency, decreased SAM and increased SAH levels were observed in the liver, but DNA methylation status in this organ was not different from controls (21
). In that animal model, SAM and SAH levels, as well as DNA methylation status in colon, did not differ between folate-deficient rats and controls. Most recently, a homozygote mutant mouse model for the enzyme methylenetetrahydrofolate reductase had significantly higher plasma homocysteine and liver SAH levels, but did not differ from wild-type mice in liver genomic DNA methylation (22
).
In the present study, the livers of homozygous mutant mice, which had higher SAM levels compared with wild-type mice had paradoxically lower DNA methylation status. The kidneys of homozygous mutant mice tended to have higher SAM levels and lower DNA methylation status compared with wild-type mice. In brain, there were no differences in SAM levels or DNA methylation status between homozygous mutant and wild-type mice. These data suggest that the response in SAM levels and DNA methylation status to elevated plasma tHcy is specific to each tissue and that high SAM levels do not necessarily increase DNA methylation.
Homozygous mutant mice had higher SAH concentrations than wild-type mice in all tissues studied, suggesting that increased plasma homocysteine does increase tissue SAH levels. However, homozygous mutant mice had significantly lower DNA methylation status than wild-type mice only in the liver. DNA methylation status in homozygous mutant mice tended to be lower in kidney and did not differ in brain compared with wild-type mice. This observation, which is similar to previous report (16
), suggests that the inhibitory effect of SAH on DNA methylation also has tissue specificity.
The SAM/SAH ratio has also been considered to be a sensitive indicator of DNA methylation (11
). In this study, homozygous mutant liver, which had lower SAM/SAH ratio than wild-type liver, had significantly lower DNA methylation status. However, homozygous mutant kidney, which had lower SAM/SAH ratio than wild-type kidney, tended to have lower methylation status. Homozygous mutant brain, which had essentially 100% lower SAM/SAH ratio than wild-type brain, did not show any significant differences in DNA methylation status. This observation suggests that the effect of SAM/SAH ratio on DNA methylation status was also tissue specific.
Marked tissue-specific differences in genomic DNA methylation have been reported in vivo (23
), and some studies suggest that hypomethylation may, in fact, be a feature of proliferating cells (24
,25
). During DNA replication, the newly synthesized strand is not methylated. After cell replication, the normal pattern of DNA methylation is catalyzed by a SAM-requiring maintenance methylase that is specific for hemimethylated sites. The maintenance methylase recognizes the 5-methylcytosine at the CpG site on the parental strand as a signal to methylate the corresponding CpG site on the daughter strand (25
). Rapidly proliferating cells, which have a relatively high proportion of hemimethylated sites in their DNA, have relatively low total genomic DNA methylation, whereas differentiated cells have relatively stable methylation patterns (26
). The liver of homozygous mutant mice had decreased DNA methylation status with increased SAM and decreased SAH concentrations, suggesting that SAH affects DNA methylation more effectively than SAM in the liver, a relatively more proliferative organ than the brain. However, the DNA methylation status of brain, a relatively less proliferating organ than liver, was not altered by the increased SAH concentration and normal SAM concentration because the CpG sites were already methylated. The methyl acceptance capacity of brain DNA was quite low compared with liver (Table 2)
. Kidney, which is also a relatively more proliferating organ than brain, tended to have lower than normal DNA methylation status, suggesting that the increased SAH concentration had more effects on DNA methylation than did the SAM concentration, which tended to be higher than controls. Our results suggest that normal or elevated SAM concentrations do not affect DNA methylation status, whereas the effect of elevated SAH concentrations on DNA methylation is tissue specific and may depend on the proliferation status of each tissue.
In conclusion, hyperhomocysteinemia induced by severe CBS deficiency increases SAH levels in liver, kidney and brain, but responses of SAM levels and DNA methylation status differed among tissues. Plasma tHcy and tissue SAM, SAH and the SAM/SAH ratio are not the sole determinants of tissue DNA methylation.
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FOOTNOTES |
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2 Any opinions, findings, conclusion, or recommendations expressed in this publication are those of the author(s) and do not necessarily reflect the view of the U.S. Department of Agriculture.
3 Supported in part by the U.S. Department of Agriculture, under agreement no. 581950–9-001 and by the Cancer Research Foundation of America (S.W.C.).
5 Abbreviations used: CBS, cystathionine ß-synthase; PCR, polymerase chain reaction; PLP, pyridoxal-5'-phosphate; SAH, S-adenosylhomocysteine; SAM, S-adenosylmethionine; tHcy, total homocysteine.
Manuscript received 28 February 2002. Initial review completed 1 April 2002. Revision accepted 20 May 2002.
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LITERATURE CITED |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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