Choline Deficiency Associated with Diethanolamine Carcinogenicity

Paul M. Newberne,1

Department of Pathology, School of Medicine, Boston University, 784 Massachusetts Ave., Boston, Massachusetts 02118

ABSTRACT

The article highlighted in this issue is "Diethanolamine Induces Hepatic Choline Deficiency in Mice" by Lois D. Lehman-McKeeman, Elizabeth A. Gamsky, Sarah M. Hicks, Jeffrey D. Vassallo, Mei-Heng Mar, and Steven H. Zeisel (pp. 38–45).

In order to place choline in proper perspective with regard to hepatocellular carcinogenesis, some sense of the history of the early events in the discovery and development of knowledge about this important methyl donor may be helpful. Choline (trimethylhydroxyethylammonium), a vitamin-like molecule, was discovered and synthesized in the mid-1860s, but its significance as a nutrient was not identified for another half-century, during studies related to the discovery of insulin by Banting and Best at the University of Toronto in 1921–1922 (Newberne, 1993Go). These investigators were able to maintain depancreatized dogs on insulin for several months, but the animals eventually developed fatty liver and died (Hershey and Soskin, 1931Go). Raw pancreas prevented the fatty liver and spared the life of the dogs, as did lecithin, an important component of pancreatic tissue. Best and Huntsman (1932) confirmed that the factor that prevented the fatty liver and demise of the dogs was choline. Intermingling of choline and its metabolites in the maintenance of methylation and methyl group metabolism, along with the difficulties in defining the input of individual members of the lipotrope family into one-carbon metabolism (choline, methionine, vitamin B12, folate), so called because they prevent the deposition of fat in the liver, slowed progress between 1932 and 1950.

Following the disclosure by Best and Huntsman (1932) that choline was a nutrient, a publication by du Vigneaud et al.(1941) described the pathway for synthesis of choline and served to energize the field of transmethylation and one-carbon metabolism. Two additional seminal articles were published, the second of which (Salmon and Copeland, 1954Go) raised choline deficiency to a level of interest that resulted in a number of laboratories initiating research in this area of carcinogenesis. This was a new research area in which something was taken out of, rather than added to, the diet (choline vs. a chemical carcinogen) in order to study neoplasia. Choline deficiency carcinogenesis helped to expand the then-growing interest in chemical carcinogenesis, and a number of investigators evaluated the promoting influence of the deficiency on traditional chemical carcinogenesis. Among many significant discoveries was the critical observation that the promoting effect was associated with rapid death and repeated waves of hepatocyte proliferation (Lombardi and Shinozuka, 1979Go), an association reported by several investigators (Newberne et al., 1982Go).

At least four enzyme-catalyzed pathways utilize choline: (1) it is oxidized to betaine aldehyde; (2) it is acetylated to form acetylcholine, the vital neurotransmitter; (3) a base-exchange pathway involves substitution of choline for serine, inositol, or ethanolamine, head groups on endogenous phospholipids; and (4) phosphorylation of the hydroxyl group of choline, catalyzed by choline kinase, the first step in the pathway to the synthesis of phosphatidylcholine.

Choline synthesis, utilization, and one-carbon metabolism interact along pathways that are designed to protect the ability to donate methyl groups to the system. At least three enzymatic pathways catalyze phosphatidylcholine biosynthesis, but only one of them, the methylation pathway, produces new choline molecules by sequentially methylating phosphatidylethanolamine (ethanolamine derived from decarboxylation of serine) using S-adenosylmethionine (SAM) as the methyl donor. This reaction is catalyzed by phosphatidylethanolamine-N-methyltransferase (PEMT), and is the major, if not the only, pathway, for de novo synthesis of choline in the mammalian hepatocyte. The other two pathways only redistribute preexisting choline molecules. The activity of PEMT is highest in the liver but also occurs in kidney, heart, brain, lung, spleen, adrenal, and erythrocytes, and appears to be contributed by a multiplicity of enzyme activities. This very brief and incomplete resumé of choline metabolism suggests that it is during these extensive and complicated interactions that growth control may be compromised in the choline-deficient liver, contributing to the neoplastic process.

Much of the research into choline deficiency carcinogenesis has focused on attempts to understand mechanisms of methyl deficiency metabolic disruptions; some progress has been made toward this goal. Important discoveries of these investigations include the following:

• Choline deficiency carcinogenesis is accompanied by decreased bioavailability of S-adenosylmethionine (SAM) and increased hepatic concentration of S-adenosylhomocysteine (SAH), a profound inhibitor of transmethylases;

• Hepatic SAM levels are decreased and SAH increased in rats administered chemical carcinogens, actions reversed by administration of choline or methione;

• Modified expression of p53 protein;

• Abnormal methylation of DNA at specific gene sites (see Christman et al., 1993Go; Newberne, 1986Go; and Shivapurkar and Poirier, 1983Go, for more detail).

Lehman-McKeeman et al. make a major contribution to a better understanding of choline deficiency carcinogenesis and, specifically, to chemical carcinogenesis induced by diethanolamine in mouse liver, by focusing on choline metabolites during dietary deficiency and comparing these data to those induced in mice by diethanolamine (DEA). The numerous metabolic changes induced by choline deficiency in the mouse liver suggest that the neoplastic process cannot be ascribed to a single factor. The rapid morphologic and biochemical disruptions in the liver cell associated with dietary choline deficiency, and the continued cell death and proliferation accompanying these disruptions, would seem to contribute appreciably to the neoplastic process; this has been strongly suggested by a number of investigators. A strength of this article is the basic data derived from the choline deficiency induced in the B6C3F1 mouse, a hybrid strain highly susceptible to chemical carcinogenesis. Untreated mice of this strain, used by the National Toxicology Program (NTP) for its chemical bioassays, sustain up to 100% liver tumors when about two years of age (see NTP 478). In addition to the B6C3F1 hybrid, some evaluations were done on the C57BL/6, one of the parent strains of the hybrid B6C3F1 mouse. This parent strain is more resistant to liver tumors than the B6C3F1, and the data on it will be useful in attempting to better understand choline deficiency and DEA carcinogenesis.

The Lehman-McKeeman studies confirmed that a short period of dietary choline deficiency, only 2 weeks, is sufficient to disrupt liver cell metabolism and result in a significant reduction in liver choline concentration, per se, as well as all choline-containing metabolites that were evaluated. While phosphocholine, the intracellular storage form of choline, decreased to the greatest extent, of particular significance were the reductions in concentrations of PC and SAM. PC, essential to synthesis of critical membrane phospholipids, derives from sequential methylation of phosphotidylethanolamine, the latter in turn derived from decarboxylation of serine. This transmethylation reaction, which appears to be the only means for de novo synthesis of choline in the mammalian hepatocyte, requires the enzyme PEMT and three moles of SAM. When SAM, the universal methyl donor reputedly involved in more than 100 critical metabolic reactions, and PC are both in short supply, the chances for disruption of metabolic processes and loss of growth control are greatly increased. Such reactions impinge on critical methylations of DNA and hyper- or hypomethylation of specific sites on genes, as noted in a number of recent publications (Christman et al., 1993Go). In addition, the increase in hepatic levels of SAH, the demethylated metabolite of SAM, is important because it is a powerful inhibitor of all methylases.

Histologic evidence of fatty liver, a hallmark of choline deficiency in rodents (Newberne, 1986Go) was not observed in either the choline-deficient or DEA-treated mice in the Lehman-McKeeman et al.study, nor was it alluded to in the chronic studies with DEA by NTP. This observation is unexplained; fatty liver has been observed in B6C3F1 choline-deficient mice in other studies. Lack of fatty liver seems to confirm that fatty liver, so commonly associated with choline deficiency in mouse and rat liver, may not, in fact, be essential to the carcinogenesis process in rodents. This is further suggested by the observation that apparently initiated nodules of hepatocytes in the choline-deficient liver exclude fat as they begin to proliferate and expand into full-blown neoplasia (Newberne et al., 1982Go).

Another observation (data not shown) in the highlighted article was a decrease in serum triglycerides, a common finding in choline-deficient rats. Importantly, it has been established that the decrease in serum triglycerides is accompanied by a concomitant increase in triglycerides in the liver and other organs and tissues; this may be the case in mice, but it apparently was not measured in the Lehman-McKeeman study. The point is that if fat (triglycerides) occupies significant space in the cell, less space and energy are left to power the multitude of metabolic processes needed for vital detoxifying events and cellular growth control mechanisms.

With essential data on the biochemistry of choline and methyl group deficiency in hand as a baseline, Lehman-McKeeman et al. evaluated liver concentrations of the same metabolites in mice exposed to DEA, with exposure similar to that used in the NTP bioassay study, except for a shorter period of time. Mice were dosed for 4 weeks with DEA, covering all exposures used in the NTP studies. Phosphocholine concentration in the liver was reduced to the greatest extent; however, all metabolites, except for SAH, but including SAM, were decreased, generally in a dose-dependent manner similar to that seen in the choline-deficient mice. SAH, the methylase inhibitor, was increased at all dose levels, particularly at the two highest doses. These changes in choline metabolite concentrations occurred despite the presence of normal amounts of choline in the diet—this may reflect a lack of capacity for uptake and utilization of choline by the hepatocyte, as demonstrated in in vitro studies with DEA—and there was no histologically identifiable fat in the livers of any of the mice. Absence of fat in the liver of choline-deficient and DEA-treated mice does not, however, detract from the argument that DEA does induce choline deficiency in the B6C3F1 mouse, as supported by biochemical measurements which equate to dietary choline deficiency. Fatty liver (presence, extent, and severity) is dependent on a number of factors (strain, age at start of diet, dietary fat quality and quantity, and other chemicals in the diet, such as antioxidants), and effects are more variable in mice than in rats. The carefully acquired analytical data of this study outweigh other factors in drawing conclusions from the DEA study. Due consideration must be accorded the fact that rats in the DEA chronic study did not develop liver tumors, even though this species is more sensitive to choline deficiency than are mice. This fact must be taken into consideration when evaluating the DEA study for evidence of human safety.

In summary, Lehman-McKeeman et al.have demonstrated that dietary choline deficiency for four weeks resulted in measurable evidence for significant decreases in choline and its metabolites in the tissues of male B6C3F1 mice. Similar biochemical modifications in choline and choline metabolite concentrations were induced in the same strain of mice by dermal application of DEA. The evidence from this study suggests that the hepatocellular carcinomas observed in mice exposed to DEA are associated with induced choline deficiency. It does not mean that DEA caused the tumors, but it does lend credence to the suggestion that the induction of hepatocellular carcinomas in the mice, associated with exposure to DEA, was likely a result of disruption of hepatocellular choline homeostasis. DEA may have simply promoted slightly the very high background incidence of tumors in this strain of mice, an action already known for choline deficiency (Lombardi and Shinozuka, 1979Go). This study represents an excellent example of identifying mechanisms of carcinogenesis by choline deficiency and by DEA and points out the constraints associated with the B6C3F1 hybrid strain of mouse as a tool for evaluation of chemicals for human safety.

NOTES

1 For correspondence via fax: (781)275-4504. E-mail: slugpoo{at}yahoo.com. Back

REFERENCES

Best, C. H., and Huntsman, M. E. (1932). The effect of the components of lecithin upon deposition of fat in the liver. J. Physiol. 75, 405–412.

Christman, J. K., Chen, M., Sheiknejad, G., Dizik, M., Abileah, A., and Wainfan, E. (1993). Methyl deficiency, DNA methylation and cancer: Studies on the reversibility of the effects of a lipotrope-deficient diet. J. Nutr. Biochem. 4, 672–680.[ISI]

du Vigneaud, V., Cohn, M., Chandler, J. P., Schenk, J. R., and Simmons, S. (1941). Pathway for the synthesis of choline. J. Biol. Chem. 140, 625–641.

Hershey, J. M., and Soskin, J. S. (1931). Substitution of lecithin for raw pancreas in the diet of the depancreatized dog. Am. J. Physiol. 98, 74–85.[Free Full Text]

Lombardi, B., and Shinozuka, H. (1979). Enhancement of 2-acetylaminofluorene liver carcinogenesis in rats fed a choline-deficient diet. Int. J. Cancer 23, 565–570.[ISI][Medline]

Newberne, P. M., de Camargo, J. L. V., and Clark, A. J. (1982). Choline deficiency, partial hepatectomy, and liver tumors in rats and mice. Toxicol. Pathol. 10, 95–106.

Newberne, P. M. (1986). Lipotropic factors and oncogenesis. In Essential Nutrients in Carcinogenesis (L. A. Poirier, P. M. Newberne, and M. W. Pariza, Eds.), pp. 223–251. Advances in Experimental Medicine and Biology, Vol. 207. Plenum Press, New York.

Newberne, P. M. (1993). The methyl-deficiency model: History, characteristics and research directions. J. Nutr. Biochem. 4, 618–624.[ISI]

Salmon, W. D., and Copeland, D. H. (1954). Liver carcinoma and related lesions in chronic choline deficiency. Ann. N.Y. Acad. Sci. 57, 664–667.[ISI]

Shivapurkar, N., and Poirier, L. A. (1983). Tissue levels of S-adenosylmethionine and S-adenosylhomocysteine in rats fed methyl-deficient, amino acid-defined diets for one to five weeks. Carcinogenesis 4, 1051–1057.[ISI][Medline]





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