Carolyn Wise

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.

Personalizing nutrigenomics research through community based participatory research and omics technologies.

Personal and public health information are often obtained from studies of large population groups. Risk factors for nutrients, toxins, genetic variation, and more recently, nutrient-gene interactions are statistical estimates of the percentage reduction in disease in the population if the risk were to be avoided or the gene variant were not present. Because individuals differ in genetic makeup, lifestyle, and dietary patterns than those individuals in the study population, these risk factors are valuable guidelines, but may not apply to individuals. Intervention studies are likewise limited by small sample sizes, short time frames to assess physiological changes, and variable experimental designs that often preclude comparative or consensus analyses. A fundamental challenge for nutrigenomics will be to develop a means to sort individuals into metabolic groups, and eventually, develop risk factors for individuals. To reach the goal of personalizing medicine and nutrition, new experimental strategies are needed for human study designs. A promising approach for more complete analyses of the interaction of genetic makeups and environment relies on community-based participatory research (CBPR) methodologies. CBPRs central focus is developing a partnership among researchers and individuals in a community that allows for more in depth lifestyle analyses but also translational research that simultaneously helps improve the health of individuals and communities. The USDA-ARS Delta Nutrition Intervention Research program exemplifies CBPR providing a foundation for expanded personalized nutrition and medicine research for communities and individuals.

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.

Cystine-Glutamate Transporter SLC7A11 Mediates Resistance to Geldanamycin but Not to 17-(Allylamino)-17-demethoxygeldanamycin

The cystine-glutamate transporter SLC7A11 has been implicated in chemoresistance, by supplying cystine to the cell for glutathione maintenance. In the NCI-60 cell panel, SLC7A11 expression shows negative correlation with growth inhibitory potency of geldanamycin but not with its analog 17-(allylamino)-17-demethoxygeldanamycin (17-AAG), which differs in the C-17 substituent in that the the methoxy moiety of geldanamycin is replaced by an amino group. Structure and potency analysis classified 18 geldanamycin analogs into two subgroups, “17-O/H” (C-17 methoxy or unsubstituted) and “17-N” (C-17 amino), showing distinct SLC7A11 correlation. We used three 17-O/H analogs and four 17-N analogs to test the role of the 17-substituents in susceptibility to SLC7A11-mediated resistance. In A549 cells, which are resistant to geldanamycin and strongly express SLC7A11, inhibition of SLC7A11 by (S)-4-carboxyphenylglycine or small interfering RNA increased sensitivity to 17-O/H, but had no effect on 17-N analogs. Ectopic expression of SLC7A11 in HepG2 cells, which are sensitive to geldanamycin and express low SLC7A11, confers resistance to geldanamycin, but not to 17-AAG. Antioxidant N-acetylcysteine, a precursor for glutathione synthesis, completely suppressed cytotoxic effects of 17-O/H but had no effect on 17-N analogs, whereas the prooxidant ascorbic acid had the opposite effect. Compared with 17-AAG, geldanamycin led to significantly more intracellular reactive oxygen species (ROS) production, which was quenched by addition of N-acetylcysteine. We conclude that SLC7A11 confers resistance selectively to 17-O/H (e.g., geldanamycin) but not to 17-N (e.g., 17-AAG) analogs partly as a result of differential dependence on ROS for cytotoxicity. Distinct mechanisms could significantly affect antitumor response and organ toxicity of these compounds in vivo.

Analytical procedure for determination of S-adenosylmethionine, S-adenosylhomocysteine, and S-adenosylethionine in same isocratic HPLC run, with a procedure for preparation and analysis of the analog S-adenosylhomocysteine sulfoxide

Abstract Virtually all methyltransferase enzymes are regulated largely by the relative levels of S-adenosylmethionine (SAM) to its metabolic product, S-adenosylhomocysteine (SAH). Ethionine is the hepatocarcinogenic antimetabolite of methionine, and has been found to produce hypomethylation of hepatic DNA when fed to rats in acute doses. The hypomethylation apparently results from the accumulation in the liver of S-adenosylethionine (SAE), the sulfur activation product of ethionine, which is a competitive inhibitor of DNA methylase. Researchers seeking to measure SAM and SAH levels by HPLC in the past have experienced numerous analytical problems because of their separation characteristics. Previous methods have either required two separate HPLC runs or used gradient elution to measure the two compounds. The method outlined here, is an accurate and precise method, that measures SAM and SAH as well as SAE in a single isocratic HPLC run. S-Adenosyl-l-homocysteine sulfoxide (SAHO), the sulfoxide of SAH is kn...

Two new ArrayTrack libraries for personalized biomedical research.

BackgroundRecent advances in high-throughput genotyping technology are paving the way for research in personalized medicine and nutrition. However, most of the genetic markers identified from association studies account for a small contribution to the total risk/benefit of the studied phenotypic trait. Testing whether the candidate genes identified by association studies are causal is critically important to the development of personalized medicine and nutrition. An efficient data mining strategy and a set of sophisticated tools are necessary to help better understand and utilize the findings from genetic association studies.DescriptionSNP (single nucleotide polymorphism) and QTL (quantitative trait locus) libraries were constructed and incorporated into ArrayTrack, with user-friendly interfaces and powerful search features. Data from several public repositories were collected in the SNP and QTL libraries and connected to other domain libraries (genes, proteins, metabolites, and pathways) in ArrayTrack. Linking the data sets within ArrayTrack allows searching of SNP and QTL data as well as their relationships to other biological molecules. The SNP library includes approximately 15 million human SNPs and their annotations, while the QTL library contains publically available QTLs identified in mouse, rat, and human. The QTL library was developed for finding the overlap between the map position of a candidate or metabolic gene and QTLs from these species. Two use cases were included to demonstrate the utility of these tools. The SNP and QTL libraries are freely available to the public through ArrayTrack at http://www.fda.gov/ArrayTrack.ConclusionsThese libraries developed in ArrayTrack contain comprehensive information on SNPs and QTLs and are further cross-linked to other libraries. Connecting domain specific knowledge is a cornerstone of systems biology strategies and allows for a better understanding of the genetic and biological context of the findings from genetic association studies.

An Improved Sample Preparation Method for the Quantitative HPLC Determination of 5-Methyl-Deoxycytidine in Animal Tissue DNA

Abstract Techniques are presented for the purification of DNA from mammalian tissues, its enzymatic hydrolysis to deoxyribonucleosides and the separation and quantification of these by high pressure liquid chromatography (HPLC). The method is used to quantify 5-niethyldeoxycytidine (5MdC) and deoxycytidine (dC) in DNA. From this data the molar %5MdC, i.e. 100 × 5MdC/(dC + 5MdC), is calculated for DNA. The precision of the method matches or exceeds that of other published HPLC methods for quantifying the %5MdC. The DNA obtained is extremely clean, and the enzymatic hydrolysis provides deoxyribonucleosides without contamination so there are no extraneous peaks in the region of interest, and peaks that are obtained have sufficient area to eliminate errors from variation in integration. The %5MdC is a quantitative and absolute measure of the genome wide DNA methylation. This method is suitable to quantify small changes in mammalian enzymatic DNA methylation due to age, diet, drugs or carcinogens.

Methamphetamine Treatment Affects Blood and Liver S-Adenosylmethionine (SAM) in Mice: Correlation with Dopamine Depletion in the Striatuma

ABSTRACT: Methamphetamine (METH) is a major drug of abuse which causes neurotoxicity by depleting dopamine, its metabolites, high‐affinity dopamine uptake sites and tyrosine hydroxylase activity in the striatum. Dopamine depletion and reduced dopamine transit are associated with depression. S‐Adenosylmethionine (SAM) is the chief methyl donor used in dopamine and other neurotransmitter metabolism in mammals. Low SAM is associated with depression and other psychological and neurological disorders in humans. SAM is used to treat depression and some other neurological and psychiatric disorders. The present study was designed to determine if single or multiple doses of METH induce alterations in blood or liver SAM in mice and if these correlate with dopamine levels in the striatum. Adult male C57 mice were injected intraperitoneally with either single (1 × 40 mg/kg) or multiple (4 × 10 mg/kg) doses of METH. Animals were sacrificed at various intervals. A single injection of METH resulted in slightly higher blood SAM levels at 4 hr. Multiple doses of METH resulted in decreased hepatic and blood SAM levels at 72 hr. Blood SAM returned to control levels by 1 wk. Published work shows that dopamine levels increase hours after a single injection of METH, whereas dopamine decreases days after multiple injections of METH. These present data clearly demonstrate that METH dosing leads to significant alterations in liver and blood SAM and that these changes in SAM levels correlate with changes in striatal dopamine levels.

A Strategy for Analyzing Gene—Nutrient Interactions in Type 2 Diabetes

Type 2 diabetes mellitus (T2DM), like all chronic diseases, results from interactions between multiple genes and multiple environmental factors. Nevertheless, many research studies focus on either nutrition or genetic factors independently of each other. The challenges of analyzing gene—nutrient interactions in T2DM are the (i) genetic heterogeneity in humans, (ii) complexity of environmental factors, particularly dietary chemicals, and (iii) diverse physiologies that produce the same apparent disease. Many of these variables are not accounted for in the design or study of T2DM or, indeed, most chronic diseases, although exceptions are noteworthy. Establishing experimental paradigms to analyze the complexity of these interactions and physiologies is challenging, but possible. This article provides a strategy to extend nutrigenomic experimental strategies to include early environmental influences that may promote adult-onset disease.