Methyl-deficient Rats Research Articles

Role of DNA damage and alterations in cytosine DNA methylation in rat liver carcinogenesis induced by a methyl-deficient diet

Currently, cancer is recognized as a disease provoked by both genetic and epigenetic events. However, the significance of early genetic and epigenetic alterations with respect to carcinogenic process in general and to liver carcinogenesis in particular remains unexplored. A lack of knowledge regarding how specific alterations during early preneoplasia may be mechanistically related to tumor formation creates a major gap in understanding the role of these genetic and epigenetic abnormalities in carcinogenesis. In the present study we investigated the contribution of DNA damage and epigenetic alterations to liver carcinogenesis induced by a methyl-deficient diet. Feeding Fisher 344 rats a methyl-deficient diet for 9 weeks resulted in DNA damage and aberrant DNA methylation. This was evidenced by an early up-regulation of the base excision DNA repair genes, accumulation of 8-oxodeoxyguanosine and 3′OH-end strand breaks in DNA, pronounced global loss of DNA methylation, and hypermethylation of CpG islands in the livers of methyl-deficient rats. These abnormalities were completely restored in the livers of rats exposed to methyl-deficiency for 9 weeks after removal of the methyl-deficient diet and re-feeding a methyl-sufficient diet. However, when rats were fed a methyl-deficient diet for 18 week and then given a methyl-sufficient diet, only DNA lesions were repaired. The methyl-sufficient diet failed to restore completely the altered DNA methylation status and prevent the progression of liver carcinogenesis. These results suggest that stable alterations in DNA methylation are a factor that promotes the progression of liver carcinogenesis. Additionally, the results indicate that epigenetic changes may be more reliable markers than DNA lesions of the carcinogenic process and carcinogen exposure.

Down‐regulation of the microRNAs miR‐34a, miR‐127, and miR‐200b in rat liver during hepatocarcinogenesis induced by a methyl‐deficient diet

Altered expression of microRNAs (miRNAs) has been reported in diverse human cancers; however, the down-regulation or up-regulation of any particular miRNAs in cancer is not sufficient to address the role of these changes in carcinogenesis. In this study, using the rat model of liver carcinogenesis induced by a methyl-deficient diet, which is relevant to the hepatocarcinogenesis in humans associated with viral hepatitis C and B infections, alcohol exposure and metabolic liver diseases, we showed that the development of hepatocellular carcinoma (HCC) is characterized by prominent early changes in expression of miRNA genes, specifically by inhibition of expression of microRNAs miR-34a, miR-127, miR-200b, and miR-16a involved in the regulation of apoptosis, cell proliferation, cell-to-cell connection, and epithelial-mesenchymal transition. The mechanistic link between these alterations in miRNAs expression and the development of HCC was confirmed by the corresponding changes in the levels of E2F3, NOTCH1, BCL6, ZFHX1B, and BCL2 proteins targeted by these miRNAs. The significance of miRNAs expression dysregulation in respect to hepatocarcinogenesis was confirmed by the persistence of these miRNAs alterations in the livers of methyl-deficient rats re-fed a methyl-adequate diet. Altogether, the early occurrence of alterations in miRNAs expression and their persistence during the entire process of hepatocarcinogenesis indicate that the dysregulation of microRNAs expression may be an important contributing factor in the development of HCC.

Influence of preconditioning-like hypoxia on the liver of developing methyl-deficient rats

Deficiency in nutritional determinants of homocysteine (HCY) metabolism, such as vitamin B(12) and folate, during pregnancy is known to influence HCY levels in the progeny, which in turn may exert adverse effects during development, including liver defects. Since short hypoxia has been shown to induce tolerance to subsequent stress in various cells including hepatocytes, and as vitamins B deficiency and hypoxic episodes may simultaneously occur in neonates, we aimed to investigate the influence of brief postnatal hypoxia (100% N(2) for 5 min) on the liver of rat pups born from dams fed a deficient regimen, i.e., depleted in vitamins B(12), B(2), folate, and choline. Four experimental groups were studied: control, hypoxia, deficiency, and hypoxia + deficiency. Although hypoxia transiently stimulated HCY catabolic pathways, it was associated with a progressive increase of hyperhomocysteinemia in deficient pups, with a fall of cystathionine beta-synthase activity at 21 days. At this stage, inducible NO synthase activity was dramatically increased and glutathione reductase decreased, specifically in the group combining hypoxia and deficiency. Also, hypoxia enhanced the deficiency-induced drop of the S-adenosylmethionine/S-adenosylhomocysteine ratio. In parallel, early exposure to the methyl-deficient regimen induced oxidative stress and led to hepatic steatosis, which was found to be more severe in pups additionally exposed to hypoxia. In conclusion, brief neonatal hypoxia may accentuate the long-term adverse effects of impaired HCY metabolism in the liver resulting from an inadequate nutritional regimen during pregnancy, and our data emphasize the importance of early factors on adult disease.

Methyl deficiency causes reduction of the methyl-CpG-binding protein, MeCP2, in rat liver.

MeCP2 is a member of a family of proteins [methyl- (cytosine-guanine)CpG-binding proteins] that bind specifically to methylated DNA and induce chromatin remodeling and gene silencing. Dietary deficiency of folate, choline and methionine causes decreased tissue S-adenosylmethionine concentrations (methyl deficiency), global DNA hypomethylation, hepatic steatosis, cirrhosis and ultimately hepatic tumorigenesis in rodents. We investigated the effects of this diet on expression of MeCP2 during pre-neoplastic transformation of liver tissue. After 9 weeks, MeCP2 mRNA level was slightly higher in methyl-deficient rats compared with replete controls, while after 36 weeks, a difference in MeCP2 mRNA level was no longer observed. In contrast, MeCP2 protein level was reduced almost 2-fold in the deficient rats compared with replete controls at both 9 and 36 weeks. Conversely, a second methyl-CpG-binding protein, MBD2, showed increased levels of both message and protein at the two time points. Low MeCP2 protein in the deficient rats was associated with a low level of the co-repressor protein, Sin3a, at 36 weeks. Moreover, a known gene target of MeCP2, the tumor suppressor gene metallothionein-I, was over-expressed in the deficient rat livers at both 9 and 36 weeks, suggesting that reduction in MeCP2 may have functional consequences. Methyl deficiency also caused an increase in the ratio of long to short variants of MeCP2 transcripts. This finding suggests that reduced MeCP2 protein level is the result of a reduced rate of translation. Reduction of MeCP2 protein expression may influence the initiation and/or progression of hepatic cancer induced by methyl deficiency and may provide a useful marker of pre-neoplastic change.

A method to estimate the percent loss of cytosine methyl groups at defined CpG sites in liver DNA from methyl-deficient rats

Dietary methyl deficiency provides an ideal in vivo model system in which to study progressive alterations in DNA methylation patterns as they occur during multistage hepatocarcinogenesis. Weanling male F344 rats were given a semipurified diet deficient in the methyl-donors choline, methionine, and folic acid for a 36-week period with sampling intervals at 3, 9, 24, and 36 weeks. Using a genomic sequencing procedure based on the PCR amplification of bisulfite-modified DNA, the methylation status of individual CpG sites within exons 6 and 7 of the p53 gene in liver samples from control and deficient rats was assessed. Treatment of denatured nuclear DNA with sodium bisulfite converts unmethylated cytosine residues to uracil, which are then amplified as thymine in the PCR reaction. In contrast, methylated cytosines are resistant to bisulfite deamination under these reaction conditions and are amplified as cytosine. In this report, we describe a novel application of automated sequencing technology to estimate the proportion of methylated cytosines present at defined CpG sites within the total population of DNA molecules extracted. Using the bisulfite conversion-PCR genomic sequencing method, we demonstrate the validity of peak height analysis of co-eluting peaks in the autosequencer electrophoregram to estimate the percent methylation at a defined CpG site. The sensitivity of this method is demonstrated by the progressive loss of methyl groups at a defined CpG site in the methyl-deficient rats after 9, 24, and 36 weeks. The application of this sequence-specific technology will allow site-specific definition of the methylation status of each CpG site within a coding sequence or promoter region and should provide new insights into mechanisms and consequences of methylation dysregulation as a result of dietary deprivation of methyl donors.

Male Rats Fed Methyl- and Folate-Deficient Diets with or without Niacin Develop Hepatic Carcinomas Associated with Decreased Tissue NAD Concentrations and Altered Poly(ADP-ribose) Polymerase Activity ,

Folate is an essential cofactor in the generation of endogenous methionine, and there is evidence that folate deficiency exacerbates the effects of a diet low in choline and methionine, including alterations in poly(ADP-ribose) polymerase (PARP) activity, an enzyme associated with DNA replication and repair. Because PARP requires NAD as its substrate, we postulated that a deficiency of both folate and niacin would enhance the development of liver cancer in rats fed a diet deficient in methionine and choline. In two experiments, rats were fed choline- and folate-deficient, low methionine diets containing either 12 or 8% casein (12% MCFD, 8% MCFD) or 6% casein and 6% gelatin with niacin (MCFD) or without niacin (MCFND) and were compared with folate-supplemented controls. Liver NAD concentrations were lower in all methyl-deficient rats after 2–17 mo. At 17 mo, NAD concentrations in other tissues of rats fed these diets were also lower than in controls. Compared with control values, liver PARP activity was enhanced in rats fed the 12% MCFD diet but was lower in MCFND-fed rats following a further reduction in liver NAD concentration. These changes in PARP activity associated with lower NAD concentrations may slow DNA repair and enhance DNA damage. Only rats fed the MCFD and MCFND diets developed hepatocarcinomas after 12–17 mo. In Experiment 2, hepatocarcinomas were found in 100% of rats fed the MCFD and MCFND diets. These preliminary results indicate that folic acid deficiency enhances tumor development. Because tumors developed in 100% of the MCFD-fed rats and because tissue concentrations of NAD in these animals were also low, further studies are needed to clearly define the role of niacin in methyl-deficient rats.

Differential sensitivity to loss of cytosine methyl groups within the hepatic p53 gene of folate/methyl deficient rats.

Dietary folate/methyl deficiency provides a unique model of endogenous hepatocarcinogenesis in which to study progressive alterations in DNA methylation patterns during tumor progression in vivo. Weanling male F344 rats were given a semi-purified diet deficient in the methyl donors choline, methionine and folic acid for a period of 9 weeks. Using a genomic sequencing procedure based on the PCR amplification of bisulfite-modified DNA, the methylation status of individual CpG sites within exons 6 and 7 of the p53 gene in liver samples from control and deficient rats was determined. Treatment of denatured nuclear DNA with sodium bisulfite quantitatively converts all cytosine residues to uracil which are then amplified as thymine in the PCR reaction. In contrast, 5-methylcytosine is resistant to bisulfite deamination under the reaction conditions and is amplified as cytosine. Automated sequencing of bisulfite-modified DNA will then elucidate the methylation status of each cytosine residue within a defined gene sequence. In addition to evaluation of the methylation status of the p53 gene, the relative activity of the DNA methyltransferase was also quantified in nuclear extracts from control and folate/methyl deficient rats. The results indicate that specific 5-methyl cytosines within the hepatic p53 gene from methyl deficient rats are resistant to demethylation despite the diet-induced decrease in S-adenosylmethionine and the increase in cell proliferation associated with this dietary intervention. Progressive demethylation was observed at other methylated cytosine residues in folate/methyl deficient rats after 9 weeks despite a paradoxical increase in DNA methyltransferase activity. The application of this sequence-specific technology will allow the definition of the methylation status of every CpG site within a coding sequence or promoter region and should provide new insights into mechanisms and consequences of methylation dysregulation during progressive multistage carcinogenesis.

Protective effect of coconut oil on renal necrosis occurring in rats fed a methyl-deficient diet.

Weanling rats fed a methyl-deficient diet develop renal necrosis with acute renal failure. The aim of this experiment was to explore further the role of coconut oil in this experimental model. Weanling Wistar male rats were fed methyl-deficient and their controls were fed methyl-supplemented diets. Coconut oil was fed at 14% and 20%, the latter concentration with and without 1% safflower oil (rich in linoleic acid); other groups received similar diets but instead of coconut oil, a mixture of hydrogenated vegetable oil and corn oil (rich in unsaturated fatty acids) was employed. Coconut oil fed at a 14% concentration did not evidence any protective outcome in relation to the renal lesions. Coconut oil at a 20% concentration showed a protective effect, mainly when the diet included safflower oil. The renal protective effect was evidenced by less or no mortality and increased survival time in the methyl-deficient rats receiving coconut oil, as well as by a reduced incidence (%) and severity of the renal lesions as evaluated by renal weight, and type (tubular and cortical necrosis or repair) and extent (grade) of the renal damage. The lack of a protective outcome when coconut oil was fed at 14%, along with the fact that in those rats receiving coconut oil at 20% the protection was greater when the diet was supplemented with 1% safflower oil, indicates that the protective effect should be attributed to the type of fatty acids coconut oil has and not to their shortage of essential fatty acids.

Liver DNA adducts in methyl-deficient rats administered a single dose of aflatoxin B1.

Using an 8 week Solt-Farber protocol with selection pressure (2-acetylaminofluorene/partial hepatectomy) applied during weeks 6 and 7, we have observed that a single oral administration of aflatoxin B1 (AFB1) to Fischer 344 rats on day 1 of the study, followed by a 3 week feeding regimen of either a methyl-deficient (CMD) or a basal (CMS) diet, results in a relative increase in hepatic preneoplastic lesions in CMD diet fed rats. It has previously been shown that a multiple dosing regimen with AFB1, started after 3 weeks of CMD diet, enhances tumor incidence. In the present study, the role of metabolic activation in the induction of preneoplastic lesions, and liver DNA adduct levels after the first dose of AFB1 in the tumorigenesis model have been investigated. AFB1-DNA adducts were determined at 2-168 h following a single non-necrogenic (100 micrograms/kg body wt) or necrogenic (600 micrograms/kg body wt) dose of AFB1 on day 1 or day 21 of a 3 week treatment with a complete basal or CMD diet. In all rats irrespective of dose, dietary treatment or time of AFB1 dosing, the patterns of adduct formation and repair did not change. In rats receiving AFB1 on day 1, total DNA adduct levels between the diet or dose groups were not significantly different, and quantitatively did not correlate with the observed increase in preneoplastic lesions, suggesting a contribution by additional factors in the initiation of these lesions. Administration of AFB1 on day 21, however, resulted in significantly reduced levels of total adducts at both dose levels in CMD diet fed rats compared to controls. Serum biochemistry data suggest that a prolonged exposure to CMD diet may cause pathological and/or biochemical alterations in hepatocytes with a resultant decrease in metabolic activation of AFB1, thus making it difficult to evaluate whether DNA damage is directly related to tumorigenesis.

Effect of Dietary Methyl Group Deficiency on One-Carbon Metabolism in Rats

Amino acid-defined diets deficient in methyl groups have been shown to result in a very high incidence of hepatocellular carcinoma. It has been suggested that this is a result of decreased levels of S-adenosylmethionine and the undermethylation of DNA. Accordingly, the enzyme glycine N-methyltransferase (GNMT, EC 2.1.1.20) may play a major role in maintaining the levels of S-adenosylmethionine in liver in response to changes in dietary methionine. The effect of methyl-deficient, amino acid-defined diets on GNMT activity and S-adenosylmethionine levels in rat liver was therefore investigated. When rats were fed a defined amino acid diet containing no choline in which homocysteine was substituted for the methionine of the control diet at an equimolar level, there was a rapid and marked decrease in growth rate in spite of the fact that the rats consumed 85% of the food eaten by control rats fed a nutritionally adequate, defined amino acid diet. The GNMT activity in livers of methyl-deficient rats decreased rapidly, but there was no difference in amount of GNMT protein as measured immunologically. In methyl-deficient rats, the levels of S-adenosylmethionine were maintained but the levels of S-adenosylhomocysteine were rapidly elevated compared to control values. These changes are consistent with the postulated role of GNMT in regulating methyl group metabolism.

Effect of Dietary Methyl Group Deficiency on Folate Metabolism in Rats

The carcinogenic effects of methyl-deficient, amino acid-defined diets have been attributed to alterations in cellular methylation reactions. These diets contain no choline, and methionine is replaced by homocysteine. Hence, all methyl groups needed for methionine biosynthesis with subsequent formation of S-adenosylmethionine and polyamines must be formed de novo utilizing folate-dependent reduction of one-carbon units. In rats fed the methyl-deficient diet, there was a marked decrease in total liver folate levels. This decrease was apparent in the levels of the individual forms of folate: 10-HCO-H4folate, 5-HCO-H4folate, 5-CH3-H4folate and H4folate. The percent of the total folate pool made up by 5-CH3-H4folate did not change, however, until after the rats had been fed the methyl-deficient diet for 4 wk, and then an increase was seen. After the methyl-deficient rats were switched to a nutritionally adequate control diet containing methionine and choline, all values rapidly reversed. Increased use of folate for methyl group biosynthesis may be responsible for the loss of folates from the liver.

An inverse correlation between hepatic ornithine decarboxylase and S-adenosylmethionine in rats

The comparative effects of the subchronic administration to rats of ethionine-supplemented and of chemically defined methyl-deficient diets on the hepatic levels of S-adenosylmethionine (SAM) and of ornithine decarboxylase (ODC), an enzyme marker of cell proliferation, were studied. Both treatments led to decreased hepatic levels of SAM and to marked increased activities in ODC. Both systems led to significant inverse correlations between ODC and SAM. In rats fed the methyl-deficient diets, hepatic levels of SAM were generally proportional to the dietary content of methionine and choline. The metabolic increases in S-adenosylmethionine decarboxylase (SAMDC) observed in the livers of methyl-deficient rats were proportional to the changes seen in ODC.