Diethylinitrosamine is a powerful hepatocarcinogen known to induce cancer in experimental animals [3, 17]. In our study, all rats given DEN developed the liver cancer, demonstrating that Sprague-Dawley rat is a suitable model in studying DEN induced liver cancer. DEN treatment was shown to significantly increase both numbers and areas of GST-p-positive foci (model group), while betaine supplementation (in all the three experimental groups) attenuated the effect of DEN. Although there was no statistical difference among the betaine treatment groups, betaine supplementation decreased the increase of GST-p in the developing of liver cancer induced by DEN. Collectively, these observations suggested that betaine can decrease the process of hepatocellular carcinoma in rats induced by DEN.
There is a strong correlation between oxidative stress and the occurrence of liver cancer [24, 25]. Enhanced lipid peroxidation and/or a dysregulated antioxidant system have been associated with liver cancer in both experimental animal models and in humans [24, 25]. Betaine acts as a methyl donor by participating in the generation of methionine from homocysteine through the catalytic action of betaine-homocysteine methyltransferase. Previous studies have shown that betaine is effective at preventing a variety of toxic injuries to the liver, such as those induced by niacin, CCl4, and hyperosmolarity [26–28]. Betaine appears to confer this protection by reducing peroxidation in the liver . In our study, we found that MDA and GST levels decreased in the 1% betaine group when compared with the model group. Although the increase in the dosage of betaine did not increase the antioxidation activity in a dose-dependent manner, our observations are consistent with the suggestion by Hayes et al. that betaine is safe and nontoxic . Betaine metabolism occurs predominantly in the liver , and DEN is able to generate acute damages in the rat liver . We found that T-AOC level was higher in the 2% and 4% betaine groups than that in either the control or the model group. These observations suggest that betaine may increase the antioxidation activity of the rat hepatocytes.
Carcinogenesis is a multi-stage process characterized by continuous changes in genotypes and phenotypes [4, 5, 7–14]. In the same way, hepatocarcinogenesis is a multistep process involving genetic and epigenetic alterations of various oncogenes, proto-oncogenes, and growth factors, as well as tumor suppressors [4, 5, 7–14]. DNA methylation is a fundamental epigenetic process that regulates not only gene transcription but also histone acetylation and chromosomal stability [11, 13, 14, 31]. DNA methylation, primarily at the C5 position of cytosine, affects gene expression during many biological processes, such as differentiation, genomic imprinting, DNA mutation, and DNA repair [11, 13, 14, 31].
Aberrant methylation of cytosine residues at CpG dinucleotides in DNA is one of the most common epigenetic changes observed in the development and progression of human cancers, including that of the liver . Shen et al. have shown that environmental factors may influence the frequency and concomitant degree of hypermethylation in multiple genes in liver cancer . They have also demonstrated that oxidative damage can directly affect DNA methylation status .
DNA methylation has been recognized as an important factor in the inactivation of tumor suppressors, such as p16, and in the activation of proto-oncogenes such as c-myc. DNA hypermethylation, usually occurring at CpG islands in promoters, is a major epigenetic mechanism in the silencing of gene expression [15, 16]. Aberrant methylation of CpG islands in the promoter region of p16 is associated with transcriptional inactivation of p16 itself. Methylation at CpG islands may inhibit gene expression, either directly by interrupting the binding of transcription activators to the promoter, or indirectly through methyl-binding proteins (MBPs) [15, 16, 32]. MBPs bind preferentially to methylated DNA sequences of a promoter and thereby silence its transcription by either competing with transcription activators for the binding sites, or by promoting histone deacetylation and chromatin remodeling that prevent transcription factors from binding to the DNA . C5 cytosine methylation at CpG sites not only significantly increases carcinogen-DNA adduct formation at CpG sites, but also affects carcinogen-DNA adduct formation at surrounding sequences .
The p16 gene encodes an inhibitor of cyclin D-dependent protein kinases. It reduces enzymatic activity of cyclin/cdk complexes, leading to aberrant phosphorylation of another tumor suppressor Rb, which, in turn, accelerates cell proliferation [34, 35]. When hypermethylation occurs in the CpG islands within the 5' flanking region of p16, its transcription is inhibited, which is a common marker of the early carcinogenic event [34, 35]. We found that compared with the rats in the control group, the rats in all the four DEN treated groups showed higher rates of hypermethylation and decreased mRNA levels of p16. This indicates that p16 hypermethylation induces p16 silencing in the rat liver cells, correlating with the development of liver cancer.
C-myc is an important regulator of various cellular processes, and has been shown to drive quiescent cells into the S phase in the absence of other mitogenic signals [5, 7, 12]. Diverse cellular functions of the c-myc oncogene are closely tied to its ability to either activate or repress gene transcription. Recently, a few studies have indicated that hypomethylation of the c-myc gene results in its over-expression in hepatocarcinogenesis, especially in the methyl-absent conditions [10, 11].
As a methyl donor, betaine is proposed to play a role in homocysteine metabolism , and provides methyl groups for the synthesis of S-adenosylmethionine (SAM) [18, 19, 37]. The requirement of SAM for cellular metabolism normally exceeds what mammals obtain through their diets. Insufficiency in SAM can be prevented through the methionine cycle that metabolizes 5'-methyltetrahydrofolate (5'-methyl-THF) and betaine . In the methionine cycle, both an increase in s-adenosyl-l-homocysteine (SAH) level and a decrease in the SAM:SAH ratio are known to reduce DNA methyltransferase activity and inhibit transmethylation reactions [18, 19, 37, 39, 42, 43]. Betaine is effective at increasing the SAM:SAH ratio and supposed to maintain normal DNA methylation patterns .
In the present study, we have, however, found that betaine had no effect on methylation of p16 or c-myc by MSP and there were no differences in c-myc methylation among the five groups of rats. It may be necessary to use more sensitive methods to quantify methylation levels. It is also possible that betaine plays a key role in maintaining complete organism-wide methylation, not the methylation of a few, specific genes. This requires further investigations.
There are two re-methylation pathways utilized by betaine and 5'-methyl-THF, which are interrelated in the methionine cycle. The limitation of one pathway increases re-methylation via the other pathway . Folic acid is the precursor of 5'-methyl-THF, and it also takes part in certain important procedures like the synthesis and reparation of DNA and RNA in addition to its effects on regulating DNA methylation. Having efficient methyl groups with three active methyls, betaine may reduce the consumption of folic acid in the methionine cycle, and it may play some part in maintaining DNA stability [42, 43]. After metabolism, betaine molecules are degraded to N5, N10-methenyl-tetra-hydrofolic acid and N5, N10-methylene-tetra-hydrofolic acid, which can be reused in the synthesis of purine and thymine. These actions can regulate the mRNA expression.
We found c-myc over-expression in the four DEN treated groups, while the enhancement was inhibited by betaine in a dose-dependent manner. Meanwhile, betaine supplementation also enhanced the down-regulation of p16 induced by DEN in a dose-dependent manner. These results suggest that betaine regulates transcription of c-myc and p16, which leads to the stability of the two genes in the body and attenuate the carcinogenic effects of DEN.