Interindividual variation in the responses of cultured human lymphocytes to exposure from DNA damaging chemical agents: Interindividual variation to carcinogen exposure

Human population variability to standardized doses of N-acetoxy-2-acetylaminofluorene (NA-AAF) and 7,12-dimenthylbenz(a)anthracene (DMBA) was determined in cultured lymphocytes by measuring (a) differential stimulation of unscheduled DNA synthesis after 1 h induction of DNA damage by 10 μM NA-AAF, (b) the level of NA-AAF induced chromosome aberrations remaining after 8 h of DNA-repair synthesis, and (c) the level of [³H]DMBA bound to DNA after 18 h incubation of resting lymphocytes in 5 μM DMBA. All 3 parameters indicated individual variation to carcinogen exposure and were correlated to the population differences in age, sex, blood pressure and mortality rates. Males always had a greater potential to accumulate DNA-damage than did females regardless of the sampled population. DNA-damage potentials increased with increasing age, blood pressure of mortality rates. There was always proportionally greater DNA-damage potentials in the males than in females. The in vitro response of mature granulocytes to a 10 μM NA-AAF dose, as estimated by [³H]thymidine incorporation from unscheduled DNA synthesis, was much lower than lymphocyte response. Nevertheless, individual variations in granulocyte NA-AAF induced unscheduled DNA synthesis paralleled the inter-individual fluctuations observed in the lymphocyte responses to NA-AAF.     

Detection and quantification of citrullinated chemokines

Background

Posttranslational deimination or citrullination by peptidylarginine deiminases (PAD) regulates the biological function of proteins and may be involved in the development of autoimmune diseases such as rheumatoid arthritis and multiple sclerosis. This posttranslational modification of arginine was recently discovered on inflammatory chemokines including CXCL8 and CXCL10, and significantly reduced their biological activity. To evaluate the importance of these modified chemokines in patients, methods for the detection and quantification of citrullinated chemokines are needed. Since citrullination only results in an increase of the protein mass with one mass unit and the loss of one positive charge, selective biochemical detection is difficult. Therefore, we developed an antibody-based method to specifically detect and quantify citrullination on a protein of interest.

Methodology/Principal Findings

First, the citrullinated proteins were chemically modified with antipyrine and 2,3-butanedione at low pH. Such selectively modified citrullines were subsequently detected and quantified by specific antibodies raised against a modified citrulline-containing peptide. The specificity of this two-step procedure was validated for citrullinated CXCL8 ([Cit⁵]CXCL8). Specific detection of [Cit⁵]CXCL8 concentrations between 1 and 50 ng/ml was possible, also in complex samples containing an excess of contaminating proteins. This novel detection method was used to evaluate the effect of lipopolysaccharide (LPS) on the citrullination of inflammatory chemokines induced in peripheral blood mononuclear cells (PBMCs) and granulocytes. LPS had no significant effect on the induction of CXCL8 citrullination in human PBMCs and granulocytes. However, granulocytes, known to contain PAD, were essential for the production of significant amounts of [Cit⁵]CXCL8.

Conclusion/Significance

The newly developed antibody-based method to specifically detect and quantify chemically modified citrullinated proteins is proven to be effective. This study furthermore demonstrates that granulocytes were essential to obtain significant levels of [Cit⁵]CXCL8. For human PBMCs and granulocytes stimulation with LPS did not affect the citrullination of CXCL8.     

DNA methylation of cord blood cell types: Applications for mixed cell birth studies

Epigenome-wide association studies of disease widely use DNA methylation measured in blood as a surrogate tissue. Cell proportions can vary between people and confound associations of exposure or outcome. An adequate reference panel for estimating cell proportions from adult whole blood for DNA methylation studies is available, but an analogous cord blood cell reference panel is not yet available. Cord blood has unique cell types and the epigenetic signatures of standard cell types may not be consistent throughout the life course. Using magnetic bead sorting, we isolated cord blood cell types (nucleated red blood cells, granulocytes, monocytes, natural killer cells, B cells, CD4⁺T cells, and CD8⁺T cells) from 17 live births at Johns Hopkins Hospital. We confirmed enrichment of the cell types using fluorescence assisted cell sorting and ran DNA from the separated cell types on the Illumina Infinium HumanMethylation450 BeadChip array. After filtering, the final analysis was on 104 samples at 429,794 probes. We compared cell type specific signatures in cord to each other and methylation at 49.2% of CpG sites on the array differed by cell type (F-test P < 10^?8). Differences between nucleated red blood cells and the remainder of the cell types were most pronounced (36.9% of CpG sites at P < 10^?8) and 99.5% of these sites were hypomethylated relative to the other cell types. We also compared the mean-centered sorted cord profiles to the available adult reference panel and observed high correlation between the overlapping cell types for granulocytes and monocytes (both r=0.74), and poor correlation for CD8⁺T cells and NK cells (both r=0.08). We further provide an algorithm for estimating cell proportions in cord blood using the newly developed cord reference panel, which estimates biologically plausible cell proportions in whole cord blood samples.     

A method for measuring the number of single- and double-strand breaks introduced into DNA molecules by the action of deoxyribonucleases.

A method is described for measuring the average number of nuclease-induced single- and double-strand breaks per DNA molecule. The procedure involves measuring the weight-average molecular weight of DNase I-digested DNA under neutral and alkaline conditions. A statistical equation is used to calculate the number of breaks per single- or double-stranded DNA molecule from the respective weight-average molecular weights. Enzymatic incorporation of³²P into the 5′-OH ends of DNase I-induced breaks gave an independent measurement of the number of breaks per DNA molecule. Results obtained by the two different methods were in good agreement. In agreement with earlier reports we find that magnesium-activated DNase catalyzes a high frequency of single-strand breaks in DNA. The frequency of double-strand breaks is low, but significantly higher than can be explained by random accumulation of single-strand breaks. Our data suggest that the frequency of double-strand scission is affected by DNase-metal ion interactions.     

Induction of deoxyribonucleic Acid synthesis in potato tuber tissue by cutting

Incorporation of ³²P-orthophosphate was found in the DNA fraction of aerobically incubated potato discs when examined by methylated albumin kieselguhr column chromatography. The estimation of DNA content of the discs was by a method developed for starchy tissues and showed that the incorporation of ³²P was due to net synthesis of DNA. The DNA content of a disc rapidly increased after a lag period of about 12 hours. The increase continued during the entire test period although at a lower rate during the later period of aging. DNA synthesis was further examined by measuring the rate of incorporation of ³H-thymidine. The striking similarity which was found between changes in the rate of DNA accumulation and in the rate of ³H-thymidine incorporation indicates that the incorporation of ³H-thymidine actually represents the net synthesis of DNA. Although the experiments with microautoradiography revealed that DNA synthesis occurred exclusively in nuclei, no signs of cell division were detected by microscopic observation. DNA synthesis in potato discs was further examined by using inhibitors of protein and RNA synthesis and was sensitive to those inhibitors. The significance of the present results is discussed in relation to the role of wounding in the induction of DNA synthesis.     

Measurement of oxidatively generated base damage to nucleic acids in cells: facts and artifacts

The search for DNA biomarkers of oxidative stress has been hampered for several decades by the lack of relevant information on base oxidation products and the challenging issue of measuring low amounts of lesions, typically a few modifications within the range 10⁶?C10⁸ normal bases. In addition and this was ignored for a long time, there is a risk of artifactual oxidation of overwhelming nucleobases during DNA extraction and subsequent workup that has led to overestimation of some base damage up to 2?C3 orders of magnitude. The main aim of the survey is to critically review the available methods that have been developed for measuring oxidatively generated base damage in nuclear and mitochondrial DNA. Among the chromatographic methods, high-performance liquid chromatography associated with tandem mass spectrometry (HPLC?CMS/MS) is the most accurate and versatile approach whereas HPLC?Celectrochemical detection (ECD) is restricted to electrochemically active modifications. These methods allow measuring several single oxidized pyrimidine and purine bases, tandem base lesions and interstrand DNA cross-links in nuclear DNA. As complementary analytical tools, enzymatic methods that associate DNA repair enzymes with either the alkaline comet assay or the alkaline elution technique are suitable for assessing low variations in the level of different classes of oxidatively generated DNA lesions. Most of the immunoassays suffer from a lack of specificity due to the occurrence of cross-reactivity with overwhelming normal bases. One major exception concerns the immunodetection of 5-hydroxymethylcytosine, produced in a relatively high yield as an epigenetic DNA modification. HPLC?CMS/MS is now recognized as the gold standard for measuring oxidized bases and nucleosides in human fluids such as urine, saliva, and plasma.     

Determination of 3J(C,P) and 3J(H,P) coupling constants in nucleotide oligomers with FIDS-HSQC

Summary A method for measuring J(C,P) and J(H,P) coupling constants is presented, based on fitting a target multiplet containing the heteronuclear coupling to a reference multiplet that lacks the heteronuclear coupling. In DNA and RNA oligonucleotides, information on backbone torsion angles can be obtained from these couplings. Experimental multiplets are obtained from ³¹P-coupled and ³¹P-decoupled ¹H, ¹³C HSQC spectra of Rp-cyclic methylphosphonate. The accuracy to which the heteronuclear coupling constants can be determined depends on the signal-to-noise ratio of the experimental data and is analyzed in detail.Dedicated to Prof. R.R. Ernst on the occasion of his 60th birthday.     

Different selenium-containing proteins in the extracellular and intracellular media of leucocytes cultivated in vitro

The purpose of this communication is to elucidate if selenium plays a role in the function of granulocytes and lymphocytes. Thus, the incorpo ration of selenium in proteins from granulocytes and lymphocytes cultured with 1ΜCi/mL radioactive Na₂ ⁷⁵SeO₃ was studied. The protein peaks containing⁷⁵Se from two columns of Heparin Sepharose CL-6B and Sephacryl S-200 HR were separated further by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis. The results showed that the incorporation of⁷⁵Se into granulocytes was about six times higher than that of lymphocytes during a 96-h cultivation, however, the GSH-Px activity in granulocytes did not change significantly. On the other hand, the GSH-Px activity of lymphocytes rose significantly after three days cultivation. These data indicated that the main chemical form of selenium in granulocytes was not GSH-Px. Results from SDS-PAGE revealed a strongly⁷⁵Se-labeled protein band with subunit molecular weight of 15 kDa in the supernatant of granulocyte homogenate. However, the main chemical forms of selenium in the culture media of granulocytes and lymphocytes were found to be selenoprotein P. The different forms of selenium-containing proteins in the intracellular and extracellular media of granulocytes indicated the different functions of these proteins.     

Diffusion of the Restriction Nuclease EcoRI along DNA

Many specific sequence DNA binding proteins locate their target sequence by first binding to DNA nonspecifically, then by linearly diffusing or hopping along DNA until either the protein dissociates from the DNA or it finds the recognition sequence. We have devised a method for measuring one-dimensional diffusion along DNA based on the ratio of the dissociation rate of protein from DNA fragments containing one specific binding site to the dissociation rate from DNA fragments containing two specific binding sites. Our extensive measurements of dissociation rates and specific-nonspecific relative binding constants of the restriction nuclease EcoRI enable us to determine the diffusion rate of nonspecifically bound protein along the DNA. By varying the distance between the two binding sites, we confirm a linear diffusion mechanism. The sliding rate is relatively insensitive to salt concentration and osmotic pressure, indicating that the protein moves smoothly along the DNA probably following the helical phosphate-sugar backbone of DNA. We calculate a diffusion coefficient for EcoRI of 3 × 10⁴ bp² s^− 1 EcoRI is able to diffuse ∼ 150 bp, on average, along the DNA in 1 s. This diffusion rate is about 2000-fold slower than the diffusion of free protein in solution. A factor of 40-50 can be accounted for by rotational friction resulting from following the helical path of the DNA backbone. Two possibilities could account for the remaining activation energy: salt bridges between the DNA and the protein are transiently broken, or the water structure at the protein-DNA interface is disrupted as the two surfaces move past each other.     

Effect of divalent ions on the minimal relaxase domain of MobA

The MobA protein encoded by plasmid R1162 plays an important role in conjugative mobilization between bacterial cells. It has two functional domains, the N-terminal relaxase domain and C-terminal primase domain. The N-terminal 186 residues (minMobA) is the minimal domain required for relaxase activity. We investigated the effects of different divalent metallic cations on minMobA activity measuring DNA binding, DNA nicking, and protein denaturation experiments. The results show that divalent cations are not required for DNA binding but are required for DNA nicking. The range of metals that function in minMobA suggests the cation role is largely structural. The most tightly binding cation is Mn²⁺, but the expressed protein shows roughly equal amounts of Mg²⁺ and Ca²⁺, both of which facilitate substrate binding and catalysis. Surprisingly, Zn²⁺ does not facilitate DNA binding nor allow nicking activity.     

Identification of polymorphonuclear leukocytes in cytologic samples for flow cytometry.

Inflammatory cells are commonly present in cytologic specimens obtained for flow cytometry, and may interfere with the analysis of epithelial cells. We have found that detergent (Triton X-100) pretreatment in the two-step acridine orange staining procedure disrupts granulocyte cell membranes to yield bare nuclei; bladder epithelial and squamous cells on the other hand are quite resistant to the detergent treatment. Being deprived of their cytoplasmic RNA, the granulocytes lose red fluorescence. Moreover, the shearing forces in the cytometer extend the multisegmented granulocyte nuclei and align them in the direction of flow. Thus, they present as elongated objects in the measuring system, giving a large DNA fluorescence pulsewidth (nuclear size). These two phenomena make it possible to identify granulocytes in the recorded data, where they are discernible from the mononucleated leukocytes and from epithelial cells. By data selection the granulocytes can be excluded, rendering epithelial cell populations more amenable to analysis. This method may make it unnecessary to remove physically leukocytes from the specimen before flow cytometry; it may also provide a way to analyze the morphology of granulocyte nuclei and to assess methods to manipulate their membrane stability. Full protection from membrane disruption is accomplished by alcohol fixation, and partial protection by 20-30% serum.     

Maillard Reaction Products Formed from l-Rhamnose and Ethylamine

A standard plasmid was constructed as a novel reference molecule for use in real-time quantitative PCR assays to verify the identity of beef, pork, chicken, mutton, and horseflesh. The plasmid contained a target domain of the cytochrome b (cyt b) gene and an artificial DNA sequence. Primers CO-F and CO-R, and probe CO-P were specifically designed to detect the artificial sequence. The calculated R² values of the standard curves (10³–10⁷ copies per reaction) for the five species ranged between 0.998 and 0.999 in the quantification analysis. The constructed plasmid provides a universal method for measuring the copy number of cyt b DNA in minced meat. This method would be a useful procedure for verifying food labels.     

Kinetics of complex formation between fluorescein mercuric acetate and DNA

The kinetics of complex formation between fluorescein mercuric acetate and heat-denatured DNA were studied by measuring the fluorescence quenching of this reagent. This quenching process involved no immeasurably rapid phase and it was shown that this reaction follows simple second-order kinetics. The rate constant at 25°C was estimated to be 2.9 × 10⁴M^?1 sec^?1 for calf-thymus DNA (42% G + C) and 1.1 × 10⁴M^?1 sec^?1 for Micrococcus lysodeikticus DNA (72% G + C). Activation parameters for this reaction were calculated from the temperature dependence of the reaction rate, and the activation entropy was found to be highly negative (?27.5 cal/mol deg for calf-thymus DNA and ?25.5 cal/mol deg for M. lysodeikticus DNA). The binding of fluorescein mercuric acetate to native DNA, which requires the opening of the double-helical structure, was also followed by measuring the absorbance change of this reagent. There was a lag phase in this binding process, and the enthalpy change for the opening step corresponded roughly to that for the opening of one base pair. These findings are discussed in relation to the results of a similar study with formaldehyde.     

A novel method for biodosimetry

Accurate methods for measuring the biological effects of radiation are critical for estimating an individual’s health risk from radiation exposure. We investigated the feasibility of using radiation-induced mutations in repetitive DNA sequences to measure genetic damage caused by radiation exposure. Most repetitive sequences are in non-coding regions of the genome and alterations in these loci are usually not deleterious. Thus, mutations in non-coding repetitive sequences might accumulate, providing a stable molecular record of DNA damage caused by all past exposures. To test this hypothesis, we screened repetitive DNA sequences to identify the loci most sensitive to radiation-induced mutations and then investigated whether these mutations were stable in vivo over time and after multiple exposures. Microsatellite repeat markers were identified that exhibited a linear dose response up to 1 Gy of 1 GeV/nucleon ⁵⁶Fe ions and ¹³⁷Cs gamma rays in mouse and human cells. Short tandem repeats on the Y chromosome and mononucleotide repeats on autosomal chromosomes exhibited significant increases in mutations at ≥ 0.5 Gy of ⁵⁶Fe ions with frequencies averaging 4.3–10.3 × 10⁻³ mutations/locus/Gy/cell, high enough for direct detection of mutations in irradiated cells. A significant increase in radiation-induced mutations in extended mononucleotide repeats was detectible in vivo in mouse blood and cheek samples 10 and 26 weeks after radiation exposure and these mutations were additive over multiple exposures. This study demonstrates the feasibility of a novel method for biodosimetry that is applicable to humans and other species. This new approach should complement existing methods of biodosimetry and might be useful for measuring radiation exposure in circumstances that are not amenable to current methods.     

In vivo distribution of cryogenically preserved guinea pig granulocytes

Granulocytes were isolated from whole blood of guinea pigs by counterflow centrifugation and labeled with [¹⁴C]diisopropylfluorophosphate ([¹⁴C]DFP). One-half of the labeled cells was injected intravenously via the femoral vein into a guinea pig, while the other half was cryogenically preserved with 5% dimethyl sulfoxide (DMSO), 6% hydroxyethyl starch (HES), and 4% human albumin, at a rate of 4 °C per minute by storage at ?80 °C and then stored for 3 days at ?80 °C. Ninety percent of the isolated granulocytes were recovered after cryogenic preservation, thawing, and washing. Aliquots before injection all produced fluorescein from fluorescein diacetate and excluded ethidium bromide. Latex ingestion was 78% and yeast ingestion was 75%. The frozen-thawed-washed-resuspended labeled granulocytes were injected into a second guinea pig. Paired animals sacrificed 35 min after injection were examined in whole-body sections for distribution of radiolabeled granulocytes to the tissues. In two pairs of animals, activity was found in the lung, liver, spleen, and kidney. The technique does not permit a distinction between fresh and cryopreserved granulocytes although there was a greater deposition of fresh cells in the liver and spleen. No activity was found in the blood of the vena cava in animals with either fresh or frozen cells. An animal injected with free [¹⁴C]DFP revealed a vascular distribution with high activity in blood, lung, and kidney, and less activity in the liver and spleen. The data indicate that radiolabeled, cryogenically preserved guinea pig granulocytes exhibited a tissue distribution qualitatively similar to fresh granulocytes, and free [¹⁴C]DFP infused without granulocytes differed qualitatively and quantitatively from fresh and cryopreserved granulocytes.     

Endogenous Oxidative DNA Damage,Aging, and Cancer

Progress in identifying the important endogenous processes damaging DNA and developing methods to assay this damage in individuals is presented. This approach may aid studies on modulation of cancer and aging.

The endogenous background level of oxidant-induced DNA damage in vivo has been assayed by measuring 8-hydroxydeoxyguanosine (oh⁸dG), thymine glycol and thymidine glycol in urine and oh⁸dG in DNA. oh⁸dG is one of about 20 adducts found on oxidizing DNA, e.g., by radiation. The level of oxidative DNA damage as measured by oh⁸dG in normal rat liver is shown to be extensive, especially in mtDNA (1/130,000 bases in nuclear DNA and 1/8,000 bases in mitochondrial DNA). We also discuss three hitherto unrecognized antioxidants in man.     

5-Ethynyl-2'-deoxycytidine as a new agent for DNA labeling: detection of proliferating cells

The labeling of newly synthesized DNA in cells to identify cell proliferation is an important experimental technique. The most accurate methods incorporate [³H]thymidine or 5-bromo-2′-deoxyruidine (BrdU) into dividing cells during S phase, which is subsequently detected by autoradiography or immunohistochemistry, directly measuring the newly synthesized DNA. Recently, a novel method was developed to detect DNA synthesis in proliferating cells based on a novel thymidine analog, 5-ethynyl-2′-deoxyuridine (EdU). EdU is incorporated into DNA and subsequently detected with a fluorescent azide via “click” chemistry. This novel technique is highly sensitive and does not require DNA denaturation. However, it was also found that EdU exhibits time-dependent inhibition effects on cell growth. Therefore, here we report a novel deoxycytidine analog, 5-ethynyl-2′-deoxycytidine (EdC), that can be used to detect DNA synthesis in vitro and in vivo at a similar sensitivity level compared with EdU. Furthermore, the EdC-induced cytotoxicity is much less than that of EdU when combined with thymidine. This will be a potential application for the long-term detection of proliferating cells.     

Ouabain,a Cardiac Glycoside,Inhibits the Fanconi Anemia/BRCA Pathway Activated by DNA Interstrand Cross-Linking Agents

Modulation of the DNA repair pathway is an emerging target for the development of anticancer drugs. DNA interstrand cross-links (ICLs), one of the most severe forms of DNA damage caused by anticancer drugs such as cisplatin and mitomycin C (MMC), activates the Fanconi anemia (FA)/BRCA DNA repair pathway. Inhibition of the FA/BRCA pathway can enhance the cytotoxic effects of ICL-inducing anticancer drugs and can reduce anticancer drug resistance. To find FA/BRCA pathway inhibitory small molecules, we established a cell-based high-content screening method for quantitating the activation of the FA/BRCA pathway by measuring FANCD2 foci on DNA lesions and then applied our method to chemical screening. Using commercial LOPAC1280 chemical library screening, ouabain was identified as a competent FA/BRCA pathway inhibitory compound. Ouabain, a member of the cardiac glycoside family, binds to and inhibits Na⁺/K⁺-ATPase and has been used to treat heart disease for many years. We observed that ouabain, as well as other cardiac glycoside family members―digitoxin and digoxin―down-regulated FANCD2 and FANCI mRNA levels, reduced monoubiquitination of FANCD2, inhibited FANCD2 foci formation on DNA lesions, and abrogated cell cycle arrest induced by MMC treatment. These inhibitory activities of ouabain required p38 MAPK and were independent of cellular Ca²⁺ ion increase or the drug uptake-inhibition effect of ouabain. Furthermore, we found that ouabain potentiated the cytotoxic effects of MMC in tumor cells. Taken together, we identified an additional effect of ouabain as a FA/BRCA pathway-inhibiting chemosensitization compound. The results of this study suggest that ouabain may serve as a chemosensitizer to ICL-inducing anticancer drugs.     

A single‐molecule approach to DNA replication in Escherichia coli cells demonstrated that DNA polymerase III is a major determinant of fork speed

The replisome catalyses DNA synthesis at a DNA replication fork. The molecular behaviour of the individual replisomes, and therefore the dynamics of replication fork movements, in growing Escherichia coli cells remains unknown. DNA combing enables a single‐molecule approach to measuring the speed of replication fork progression in cells pulse‐labelled with thymidine analogues. We constructed a new thymidine‐requiring strain, eCOMB (E. coli for combing), that rapidly and sufficiently incorporates the analogues into newly synthesized DNA chains for the DNA‐combing method. In combing experiments with eCOMB, we found the speed of most replication forks in the cells to be within the narrow range of 550–750 nt s^?1 and the average speed to be 653 ± 9 nt s^?1 (± SEM). We also found the average speed of the replication fork to be only 264 ± 9 nt s^?1 in a dnaE173‐eCOMB strain producing a mutant‐type of the replicative DNA polymerase III (Pol III) with a chain elongation rate (300 nt s^?1) much lower than that of the wild‐type Pol III (900 nt s^?1). This indicates that the speed of chain elongation by Pol III is a major determinant of replication fork speed in E. coli cells.     

Global methylation profiles in DNA from different blood cell types

DNA methylation measured in white blood cell DNA is increasingly being used as in studies of cancer susceptibility. However, little is known about the correlation between different assays to measure global methylation and whether the source of DNA matters when examining methylation profiles in different blood cell types. Using information from 620 women, 217 and 403 women with DNA available from granulocytes (Gran), and total white blood cells (WBC), respectively, and 48 women with DNA available from four different sources (WBC, Gran, mononuclear (MN), and lymphoblastoid cell lines (LCL)), we compared DNA methylation for three repetitive elements (LINE1, Sat2, Alu) by MethyLight, luminometric methylation assay (LUMA), and [³H]-methyl acceptance assay. For four of the five assays, DNA methylation levels measured in Gran were not correlated with methylation in LBC, MN, or WBC; the exception was Sat2. DNA methylation in LCL was correlated with methylation in MN and WBC for the [³H]-methyl acceptance, LINE1, and Alu assays. Methylation in MN was correlated with methylation in WBC for the [³H]-methyl acceptance and LUMA assays. When we compared the five assays to each other by source of DNA, we observed statistically significant positive correlations ranging from 0.3-0.7 for each cell type with one exception (Sat2 and Alu in MN). . Among the 620 women stratified by DNA source, correlations among assays were highest for the three repetitive elements (range 0.39-0.64). Results from the LUMA assay were modestly correlated with LINE1 (0.18-0.20). These results suggest that both assay and source of DNA are critical components in the interpretation of global DNA methylation patterns from WBC.