Lionel A. Poirier

The Effects of Diet, Genetics and Chemicals on Toxicity and Aberrant DNA Methylation: an Introduction

In the early 1930s, the group of Banting and Best showed that the choline moiety of lecithin was responsible for the prevention of the fatty livers produced in pancreatectomized dogs treated with insulin. This was the first study linking abnormal methyl metabolism with disease. Since then, deficiencies of each of the four essential dietary sources of methyl groups (choline, methionine, vitamin B-12 and folic acid) have been associated with increased risk of a number of diseases. Choline-deficient diets were shown to enhance liver tumor formation in rats, and such diets frequently were found to lead to atherosclerosis. Although methionine deficiency per se was not extensively studied in vivo, its metabolic antagonist ethionine did cause liver cancer and pancreatic toxicity in rodents. Deficiencies of vitamin B-12 and of folic acid have long been shown to cause neurological disturbances and birth defects both in humans and in experimental animals. In 1969 inborn errors of metabolism leading to the accumulation of the demethylated metabolite of methionine, homocysteine, were proposed as contributing to the early onset of atherosclerosis. Before 1990, numerous studies described the abnormal methylation of DNA in tumors and transformed cells. Less frequently investigated, however, were the exogenous and endogenous agents leading to such abnormal methylation. These included genetic variants among rodent strains and the methyl-deficient diets that caused liver cancer. In addition, several chemicals, particularly carcinogens, were shown to alter DNA methylation. The possible links between chemically induced alterations in DNA methylation and development of other diseases were little explored. However, by 1990, a chain of causality had been established in experimental carcinogenesis linking dietary methyl deficiency with methyl insufficiency in vivo, as well as with the abnormal methylation of DNA and of specific genes. Also during this period, the diminished activity of the enzyme methylenetetrahydrofolate reductase (EC 1.5.1.20), which is responsible for the actual de novo synthesis of methyl groups, was shown to be associated with increased risk of developing atherosclerosis, neurological disorders and birth defects. The exponential rise in studies on methyl metabolism and DNA methylation since then enables us to examine here the extent to which the mechanisms by which abnormal methylation processes seem to exert their toxic effects in one disease may be applicable to other pathologies.

Antagonism by essential divalent metals and amino acids of nickel(II)-DNA binding in vitro.

In vitro binding of nickel(II) to DNA and the effects of divalent essential metals calcium, magnesium, manganese, copper, and zinc, and of amino acids histidine, cysteine, glutamine, arginine, lysine, alanine, and glycine upon that binding were investigated. Samples of 0.156 mg of calf thymus DNA (0.078 mg/ml in 5 mM ammonium acetate buffer, pH = 7.4) were incubated for 1 hr at 24 degrees C with various concentrations of nickel(II)acetate labeled with 63Ni (0.1 to 250 microM) in the absence or presence of 50 microM concentrations of the essential metal acetates, or with 100 microM concentrations of the amino acids. Free and DNA-bound nickel(II) fractions were separated by gel filtration on Sephadex G-25 and quantified by liquid scintillation counting. Scatchard analysis revealed more than two types of nickel(II)-binding sites and a positive cooperativity of binding at the bound-Ni concentrations below 0.35 microM. The high-affinity nickel(II)-binding sites at DNA were identified as phosphate groups. Their binding capacity equalled 0.043 mumol/mg DNA (approx. 1 mol Ni/70 mol of DNA bases). The apparent dissociation constant of nickel(II) from the high-affinity sites was 5.35 microM. Double reciprocal plots showed the essential divalent metals to be competitive antagonists of nickel(II)-binding to the high-affinity sites, ranking Mg(II) greater than or equal to Mn(II) greater than or equal to Ca(II) greater than or equal to Cu(II) = Zn(II). Similarly, the amino acids antagonized nickel binding to DNA with a relative strength of His greater than Gln greater than or equal to His/Cys greater than Arg greater than Cys greater than or equal to Gly = Ala greater than or equal to Lys. The strongest inhibitors of nickel(II)-DNA binding in vitro appear to be magnesium and manganese, i.e., the same metals that are capable of attenuating nickel carcinogenicity in vivo.

Measuring S-adenosylmethionine in whole blood, red blood cells and cultured cells using a fast preparation method and high-performance liquid chromatography

The physiological methyl donor S-adenosylmethionine (SAM) plays a key role in the maintenance of human health and in the prevention of disease. A convenient clinical test for blood SAM does not exist, even though blood SAM is increasingly seen as an important indicator of health. We have developed a simple procedure to extract SAM from small amounts of blood or cells. The extracted SAM is then measured by high-performance liquid chromatography (HPLC). This measurement is sensitive, precise and uses as little as 200 microliters of blood or 0.5-10(6) cultured cells per determination. SAM, as tested with this method, under acidic conditions, is stable for hours and can be frozen for later analysis. The method has been used to show that blood SAM varies with species, sex and treatment. We have also measured the SAM levels in cultured cells, and have been able to detect wide variations depending upon treatments administered during the growth of those cells. In conclusion, this is a very rapid and easy method to measure SAM in biological fluids and cell culture and which could be adapted to the clinical setting.

S-adenosyl-L-methionine is able to reverse micronucleus formation induced by sodium arsenite and other cytoskeleton disrupting agents in cultured human cells.

Deficiencies of folic acid and methionine, two of the major components of the methyl metabolism, correlate with an increment of chromosome breaks and micronuclei. It has been proposed that these effects may arise from a decrease of S-adenosyl-L-methionine (SAM), the universal methyl donor. Some xenobiotics, such as arsenic, originate a reduction of SAM levels, and this is believed to alter some methylation processes (e.g. DNA methylation). The aim of the present work was to analyze the effects of exogenous SAM on the micronucleus (MN) frequency induced by sodium arsenite in human lymphocytes treated in vitro and to investigate whether these effects are related to DNA methylation. Results showed a reduction in the MN frequency in cultures treated with sodium arsenite and SAM compared to those treated with arsenite alone. To understand the mechanism by which SAM reduced the number of micronucleated cells, its effect on MN induced by other xenobiotics was also analyzed. Results showed that SAM did not have any effect on the increase in MN frequency caused by alkylating (mitomycin C or cisplatin) or demethylating agents (5-azacytidine, hydralazine, ethionine and procainamide), but it reduced the number of micronucleated cells in those treated with agents that inhibit microtubule polymerization (albendazole sulphoxide and colcemid). Since albendazole sulphoxide and colcemid inhibit microtubule polymerization, we decided to evaluate the effect of SAM on microtubule integrity. Data obtained from these evaluations showed that sodium arsenite, albendazole sulphoxide, and colcemid affect the integrity and organization of microtubules and that these effects are significantly reduced when cultures were treated at the same time with SAM. The data taken all together point out that the positive effects of SAM could be due to its ability to protect microtubules through an unknown mechanism.

Ornithine decarboxylase induction and polyamine levels in the kidney of estradiol-treated castrated male rats

Abstract Ornithine decarboxylase (ODC), S-adenosylmethionine decarboxylase (SAMDC), and thymidine kinase (TK) activities and polyamine concentrations on the kidneys of male castrated rats were studied following sc injection of estradiol. Estradiol caused an 11-fold increase in ODC activity 24 hours after administration. SAMDC activity doubled but TK activity decreased by two-thirds 2 days after estradiol treatment. The concentrations of polyamines, especially putrescine, showed sharp elevations 2 days following estradiol treatment, 1 day after the peak of ODC activity. The increase in ODC activity was suppressed by cycloheximide and by actinomycin D. Estradiol and diethylstilbestrol (DES), but not progesterone increased ODC activity. Estradiol suppressed ODC activities of liver, thymus, adrenal glands, testes and prostate. A specific estradiol-binding protein was demonstrated in the rat kidney. The dissociation constant (Kd) was 1.64 × 10−10 M and numbers of binding sites were 31 fmoles/mg protein. Correlation between the binding of estradiol to the cytosol protein and elevation of ODC by estradiol was observed.

Increasing levels of dietary homocystine with carotid endarterectomy produced proportionate increases in plasma homocysteine and intimal hyperplasia.

PURPOSE The role that homocysteine may play in post-carotid endarterectomy (CEA) restenosis due to intimal hyperplasia is not well understood. This study was designed to investigate the effects of different levels of dietary homocystine on: (1) plasma homocysteine; (2) post-CEA intimal hyperplasia; and (3) levels of the methyl donor S-adenosylmethionine (SAM) and its counterpart S-adenosylhomocysteine (SAH) in the homocysteine pathway. METHODS Male rats were fed specialized diets for 2 weeks pre- and post-CEA. Groups included control (0 homocystine added, n=9), 1.5 (1.5 g/kg homocystine added, n=10), 3.0 (3.0 g/kg homocystine added, n=9), and 4.5 (4.5 g/kg homocystine added, n=11). The rats underwent a surgical carotid endarterectomy. Endpoints included; plasma homocysteine, intimal hyperplasia, replicative index using with alpha-SM actin and BrdU, hepatic SAM levels, SAH levels, and the hepatic activities of methylenetetrahydrofolate reductase (MTHFR) and cystathionine beta-synthase (CBS). RESULTS Increasing dietary homocystine produced a proportionate increase in plasma homocysteine and an increase in intimal hyperplasia. Regression analysis of plasma homocysteine levels and intimal hyperplasia showed a significant correlation (r=0.71,P=0.003). Plasma homocysteine levels above 15 microM were associated with significant increases in intimal hyperplasia above 6.5% (P=0.04). Elevation of plasma homocysteine levels to moderate levels (5-25 microM) resulted in significant post-CEA intimal hyperplasia. Cellular analysis of the area of intimal hyperplasia in all diet groups showed comparable amounts of cells positive for alpha-SM actin. However, with increasing levels of dietary homocystine and plasma homocysteine there was an increase in replicative index (P<0.001) as determined by BrdU staining. Increasing dietary homocystine increased plasma homocysteine and was followed by increases in the replicative index thus producing increased intimal hyperplasia and lumenal stenosis. In hepatic measurements the 1.5 and 3.0 g/kg homocystine diets caused: increased liver activity of MTHFR (P=0.03) and decreased hepatic levels of SAM, SAH and SAM/SAH ratios compared to controls. Homocystine treatment did not cause significant alterations in CBS levels (P=0.992). These studies also showed no correlation of the MTHFR and CBS enzymes with plasma homocysteine levels or intimal hyperplasia. However, hepatic levels of SAM showed significant negative correlations with plasma homocysteine (r=-0.58; P=0.006) and with BrdU percentages of cellular proliferation (r=-0.69; P=0.06). CONCLUSION The degree of post-CEA intimal hyperplasia in a rat model is directly related to the plasma level of homocysteine. The hyperplastic effects of homocysteine may be mediated in part by a physiological insufficiency of methyl donors as shown by decreases in SAM. Thus, increasing levels of plasma homocysteine enhanced and accelerated the smooth muscle cell response after CEA which led to increased intimal hyperplasia and lumenal stenosis.

Increased expression of hepatic DNA methyltransferase in smokers

The DNA methyltransferase enzyme (DNA MTase) catalyzes DNA methylation at cytosines in CpG dinucleotides. 5-Methylcytosine modification of DNA is important in gene regulation, DNA replication, chromatin organization and disease. Increased levels of DNA MTase have been associated with the initiation and promotion of cancer. This study was conducted to assess whether cigarette smoking and other factors, such as age and gender, influence DNA MTase expression in nontumorous tissue. DNA MTase was significantly (p<0.05) higher in samples from cigarette smokers; the mean level of DNA MTase mRNA was almost 2-fold higher in these samples than in those from nonsmokers. Levels of DNA MTase mRNA were higher in samples from females than in those from males, but the difference was not statistically significant. Age was not associated with DNA MTase levels. Increased levels of DNA MTase in individuals who smoke may indicate a greater susceptibility to the risk of cancer since increased levels of this enzyme are found in cancer cell lines and human tumors. The results of this study suggest that further investigations of increased expression of this enzyme as a predisposing factor for cancer susceptibility are needed.

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.

Reduced cadmium-induced cytotoxicity in cultured liver cells following 5-azacytidine pretreatment☆

Recent work indicated that administration of the pyrimidine analog 5-azacytidine (AZA), either to cells in culture or to rats, results in an enhancement of expression of the metallothionein (MT) gene. Since MT is thought to play a central role in the detoxification of cadmium, the present study was designed to assess the effect of AZA pretreatment on cadmium cytotoxicity. Cultured rat liver cells (TRL 1215) in log phase of growth were first exposed to AZA (8 microM). Forty-eight hours later, cadmium (10 microM) was added. MT concentrations were then measured 24 hr after the addition of cadmium. A modest increase in MT amounts over control (1.7-fold) was detected after AZA treatment alone. Cadmium alone resulted in a 10-fold increase in MT concentrations. The combination of AZA pretreatment followed by cadmium exposure caused a 23-fold increase in MT concentrations over control. Treatment with the DNA synthesis inhibitor hydroxyurea (HU) eliminated the enhancing effect of AZA pretreatment on cadmium induction of MT, indicating that cell division is required. AZA-pretreated cells were also harvested and incubated in suspension with cadmium (250 microM, 37 degrees C) for 0 to 90 min. After incubation intracellular and extracellular fluids were separated by centrifugation through an oil layer. AZA-pretreated cells showed marked reductions in cadmium-induced cytotoxicity as reflected by reduced intracellular potassium loss, glutamic-oxaloacetic transaminase loss, and lipid peroxidation (assessed by thiobarbituric acid reactants) following cadmium exposure. AZA pretreatment had no effect on the cellular uptake of cadmium. Results suggest that AZA pretreatment induces tolerance to cadmium cytotoxicity which appears to be due to an increased capacity to synthesize MT rather than high quantities of preexisting MT at the time of cadmium exposure.

Effects of essential divalent metals on carcinogenicity and metabolism of nickel and cadmium

Interactions between the physiologically essential metals calcium, magnesium, and zinc and the carcinogenic metals nickel and cadmium were investigated to help elucidate the mechanisms of action of the carcinogenic metals. Bioassay studies revealed several significant findings, including: (1) the ability of magnesium and calcium to inhibit nickel-induced elevation of pulmonary adenoma incidence in strain A mice; (2) the ability of magnesium, but not of calcium, to prevent cadmium-induced subcutaneous sarcoma formation; and (3) the ability of magnesium, but not of calcium, to inhibit nickel-induced muscle tumor formation. Biochemical studies indicated a direct relationship between the antitumorigenic potential of magnesium and the capacity of this metal to: (1) inhibit nickel and cadmium uptake by the target tissues in vivo; (2) inhibit nickel-induced disturbances in DNA synthesis in vivo; (3) inhibit nuclear and cytosolic uptake of nickel by the target tissue cells in vivo; and (4) inhibit nickel and cadmium binding to DNA in vitro. Calcium, which in most cases did not prevent carcinogenesis, had no consistent influence on the uptake of carcinogenic metals or their biochemical effects in the target tissues. Magnesium and zinc, but not calcium, were also found to attenuate the acute toxic effects of nickel, indicating a possible correlation between prevention of acute effects and reduction in tumorigenicity. Zinc, which antagonizes cadmium tumorigenicity in the rat testis, was found to reduce markedly cadmium uptake into isolated testicular interstitial cells. Also, zinc was found to inhibit strongly cadmium binding to DNA in vitro.