Analysis of polymerase chain reaction-amplified DNA products by mass spectrometry using matrix-assisted laser desorption and electrospray: current status.

Recent advances in molecular biology are making it possible to diagnose genetic diseases and identify pathogens through the analysis of DNA. As clinical applications for molecular diagnosis increase, rapid, reliable methods for determination of DNA size will be needed. Mass spectrometry offers the potential of analyzing amplified DNA quickly and reliably, without the need for gel-based separation and sample labeling steps that are conventionally employed. Both electrospray ionization and matrix-assisted laser desorption/ionization have been evaluated for the size analysis of DNA using both synthetic oligonucleotides and PCR-amplified samples corresponding to bases 1626 to 1701 of the cystic fibrosis transmembrane conductance regulator gene. Both technologies have been demonstrated to have mass range and sensitivity required for the analysis of PCR-amplified DNA in this size range using minimal sample preparation. Steps required to incorporate either ionization technique into a reliable analytical scheme for the rapid, routine analysis of DNA are outlined.

Measurement of DNA adducts using surface-enhanced Raman spectroscopy.

Hazardous pollutants emitted from energy-related technologies, chemical industries, or waste materials are of increasing public concern because of their potential adverse health effects. Many pollutants have chemical groups of toxicological importance that can be characterized and detected by Raman spectroscopy. Raman spectroscopy, however, has not been widely used in trace organic detection, even though the information contained in a Raman spectrum is valuable for chemical identification. One limitation of conventional Raman spectroscopy is its low sensitivity, which often necessitates the use of powerful and costly laser sources for sample excitation. Raman spectroscopists have recently been able to analyze dilute biological samples as a result of enhancements in the Raman scattering cross section by factors up to 10(10) when a compound is adsorbed on or near a special electron-conducting surface. These spectacular enhancement factors of the normally weak Raman scattering process help overcome the low sensitivity of Raman spectroscopy through a combination of electromagnetic and chemical interactions between the analyte molecule and the surface. The technique associated with this phenomenon is known as surface-enhanced Raman scattering spectroscopy (SERS). The special conductive surface responsible for the scattering enhancement is referred to as a SERS substrate. For the past few years we have developed the SERS technique, using practical SERS-active substrate materials based on silver-coated microspheres deposited on glass. A wide variety of biomarkers have been investigated, including benzo[a]pyrene, dibenz[a,h]anthracene epoxides, 1,N9-ethenoadenine, 3,N4-ethenocytosine, and other substances. These biomarkers were measured at nanogram and subnanogram levels. The experimental results are of great analytical interest, since these chemicals are difficult to detect by other techniques, such as luminescence spectroscopy, because of the weak luminescence quantum yields of these DNA adducts. In this paper the potential usefulness of the SERS technique for assessing environmental and health effects from human exposure to toxic pollutants is demonstrated.

Comparison of supernatant-and ribosome-bound rat liver tRNA.

Transfer RNAs were separated into a ribosome bound fraction and a supernatant (cytoplasmic) fraction. The nucleoside composition, 2-dimensional PAGE pattern and in vivo labeling were compared. 12 minor nucleosides were identified by HPLC. In general, the minor nucleosides, especially N2-methyl-guanidine and ribothymidine, were higher in the ribosome-bound fraction. The PAGE patterns were similar but there were quantitative and qualitative differences among the smaller spots. In vivo labeling by 32P showed that new tRNA goes preferentially to the ribosome but mixing does occur. The results suggest the existence of two compartments of tRNA.

DNA adduct formation by 12 chemicals with populations potentially suitable for molecular epidemiological studies.

DNA adduct formation, route of absorption, metabolism and chemistry of 12 hazardous chemicals are reviewed. Methods for adduct detection are also reviewed and approaches to sensitivity and specificity are identified. The selection of these 12 chemicals from the Environmental Protection Agency list of genotoxic chemicals was based on the availability of information and on the availability of populations potentially suitable for molecular epidemiological study. The 12 chemicals include ethylene oxide, styrene, vinyl chloride, epichlorohydrin, propylene oxide, 4,4'-methylenebis-2-chloroaniline, benzidine, benzidine dyes (Direct Blue 6, Direct Black 38 and Direct Brown 95), acrylonitrile and benzyl chloride. While some of these chemicals (styrene and benzyl chloride, possibly Direct Blue 6) give rise to unique DNA adducts, others do not. Potentially confounding factors include mixed exposures in the work place, as well the formation of common DNA adducts. Additional research needs are identified.

Direct labeling of DNA-adducts formed from carcinogenic diol-epoxides with a fluorescent reporter compound specific for the cis vic-diol group.

The fluorescent reporter group, N-(5-FLUORESCEINYL),N'-(3-BORONATOPHENYL)THIOUREA (FABA) has been synthesized. This boronate group makes the reporter specific for cis vic-diols. The reporter group is bound to DNA-adducts formed from the reaction of calf-thymus DNA and benzanthracene trans-10,11-dihydrodiol,8,9-epoxide (anti), benzo(a)anthracene-trans-3,4-dihydrodiol-1,2-epoxide (anti) but is not bound to 3-methylcholanthrene-11,12 dihydro-epoxide. Femtomole quantities of adduct may be detected.

Rapid oxidative-elimination of the terminal base from model deoxynucleotide esters.

The 5'-isopropyl esters of 2'-deoxycytidylate, 2'-deoxyadenylate, 2'-deoxyguanylate and 2'-deoxythymidylate were synthesized and used as model substrates to demonstrate that the 3'-terminal deoxynucleoside can be rapidly removed by combined 3'-hydroxyl oxidation and beta-elimination. Trifluoroacetyloxy-dimethylsulfonium ion oxidation of the free 3'-hydroxyl group gave almost 80% yields of the free purine and pyrimidine bases in less than 5 minutes reaction at -80 degrees C.

Persistence of benzo[a]pyrene and 7,8-dihydro-7,8-dihydroxybenzo[a] pyrene in Fischer 344 rats: time distribution of total metabolites in blood, urine and feces.

A comparison of the rates of elimination of [3H]benzo[a] pyrene (BaP) and 7,8-dihydro-7,8-diol-[3H]benzo[a]pyrene (BPD), after subcutaneous injection into Fischer 344 rats, shows they are both eliminated at about the same rates and with the same pattern over at least 7 days post-exposure. The end-rate of combined urinary and fecal excretion was approximately 40 nmol/day. About 20% of the injected BaP and approximately 3% of the injected BPD remained at the site of injection for at least 9 days. The remainder was distributed throughout the animal. If the rate of excretion continued at the observed steady-state rates, the BaP and BPD could persist for up to 40 days for each milligram of injected substance. The concentration of excretion products were highest during day 1 and day 2 following exposure, decreased exponentially to a concentration of approximately 0.5 microM (mixed metabolites) by day 5 following exposure, and then continued to be excreted at that rate. Feces contained the highest total amounts of radioactivity, which were approximately 2- to 4-fold higher than the amounts in urine and approximately 15- to 50-fold higher than in total blood. The conversion of organic 3H to 3H2O during the experimental period indicates that whole-body phenol(quinone) formation was significant for BaP metabolism, but was much less for BPD metabolism. When BaP was injected, both blood and urine contained water-soluble, volatile tritium counts (3H2O). Injection of BPD resulted in volatile 3H2O in urine but not in blood. The persistence of BaP and BPD metabolites in skin, blood, urine and feces compartments indicates there is a substantial reservoir of the chemical(s) that could be used to replenish repaired or discarded DNA adducts.

Effects of toxic chemicals on the release of pyrimidine compounds in cell culture.

Exposure of hamster embryo cells and BF lymphoblastoid cells to 18 known toxic substances and four nominally nontoxic substances results in the release of pyrimidines (and their nucleosides) into the culture medium. The extent of release is dependent on the specific chemical and the specific cells present in the assay. BF cells are not affected by exposure to benzo(a)pyrene, while the hamster embryo cells exhibit enhanced excretion on exposure to benzo(a)pyrene. This difference in response may be due to the difference in endogenous aryl hydrocarbon hydroxylase (BaP) activity. In contrast, diethylstilbestrol, which is metabolized by a peroxidase-mediated enzyme system, causes enhanced excretion in both cell types. Direct alkylating agents and Ni(+2) salts also cause enhanced excretion in both cell types. We have used concentrations of chemicals that give a 5% enhanced excretion as the criterion of low-dose response. Within the range of concentrations tested, chromate induces enhanced excretion in BF cells but not the HEC cells, and Pb(+2) induces enhanced excretion in HEC cells but not the BF cells. Benzene, dimethylnitrosamine, and Mg(+2) did not affect either cell type. 7,12-Dimethylbenzo(a)anthracene, anthracene, benzo(a)anthracene, phenylazoaniline, N-methyl, N-nitroso, N'-nitroguanidine, dioxane, and pyrene cause enhanced excretion in the hamster embryo cells while benzo(e)pyrene, ZnSO4 and cholesterol do not cause enhanced excretion in the hamster embryo cells. Of those chemicals causing enhanced excretion, the concentration range bracketing 5% enhanced excretion approximated low-dose exposures reported to result in toxic responses like cancer, teratogenesis or pulmonary disease.

Toxic response of hamster embryo cells on exposure to mixtures of Ni2+ and benzo(a)pyrene.

Exposure of hamster embryo cells (HEC) to mixtures of benzo(a) pyrene and soluble Ni2+ neutral salts elicits additive responses in the enhanced nucleoside excretion assay. Ni2+ shows no significant excretion response until concentrations near 10 microM are reached. Addition of 0.4 micromolar benzo(a)pyrene and 30 microM, or 325 microM Ni2+ to HEC cultures results in total excretion equal to the sum of excretions induced by the individual chemicals. These observations suggest that excretion of pyrimidines may be a useful measure of biological dosimetry.

Batch separation of uracil derivatives from urine.

Commercial mixed-bed deionizing resins have been modified by conversion to the sulfonic acid and acetate forms and used to prepare extracts of urine that contains uracil derivatives (pKa greater than 8). A rapid batch extraction process, requiring less than 15 min is described for the first time that can be used either for rapid spectrophotometric screening of altered levels of these uracil derivatives or for accurate measurement of uracil and pseudouridine by high-performance liquid chromatography on reversed-phase columns using 0.002 M ammonium formate (pH 6) in water as the solvent. The spectrophotometric assay is based on the spectral shift of uracil derivatives when neutral aqueous solutions are made alkaline. After the batch extraction, the factor for conversion of absorbancy change to nmol is 0.0046 per nmol/ml for uracil and 0.0035 per nmol/ml for pseudouridine. Pseudouridine levels in two test urines were found to be 24 and 19.3 nmol/mumol of creatinine, which are consistent with published values of 17.6-24.

Aryl hydrocarbon hydroxylase tissue-specific activities: evidence for baseline levels in mammalian tissues.

The tissue-specific activities (units per gram tissue) of arylhydrocarbon hydroxylase benzo[a]pyrene [AHH(BaP)] (EC 1.14.14.2) in human, mouse, rat, and hamster have been reviewed. Three categories of AHH activities are defined: baseline values from tissues that have been protected from adventitious exposures to AHH inducers; background levels from tissues where there have been no overt measures to protect against exposure; and induced levels resulting from overt exposure to chemical inducers. Evidence that the baseline category exists is derived from the observations that an upper limit of AHH tissue-specific activity of about 1.5 nmol/h . g tissue occurs in human placenta, human foreskin, lymphocyte, and epitheliod and fibroblastoid cell lines; mouse lung and liver; rat fetal liver, and noninducible rat cell lines from lung, liver, embryo kidney, and adrenals; and hamster kidney. The collected values for nonexposed tissues range from 0.02 nmol/h . g to values less than 1.5 nmol/h . g. The most consistent observation of this type was from human placental material from nonsmoking mothers. Animals raised under standard laboratory conditions without special dietary precautions show background AHH activities that range from 2 nmol/h . g to 200 nmol/h . g in portal of entry tissues such as liver, lung, and intestines. Almost all tissue samples showed induced AHH levels of up to 500 nmol/h . g when those tissues were overtly exposed to substances containing chemical inducers of AHH. Measurements of placental AHH from smoking mothers showed that more than 95% of those samples had AHH values exceeding 2.5 nmol/h . g. This natural bimodal distribution of AHH activities, across species and in different tissues, of baseline values of less than 1.5 nmol/h . g and background or induced AHH activities with values greater than 1.5 nmol/h . g, may provide a reference set of values for use in quantification of the role of AHH in the induction of disease.

RNA turnover in cultured hamster embryo cells: identification of modified nucleoside end products.

Fourteen methylated nucleosides (N-2-dimethylGuo, N-2-methylGuo, N-1-methylGuo, N-5-methylUrd, N-3-methylUrd, N-1-methylAdo, N-3-methylCyd, N-5-methylCyd, N-1-methyllno, 2'-Omethyl-Cyd, 2'-O-methylUrd, 2'-0-methylGuo, 2'-0-methyllno, and thymidine) and one methylated base (m7Gua) have been identified as normal excretion products of cultured hamster embryo cells. The methylated nucleosides are excreted in the culture media subsequent to RNA turnover. The excretion pattern of the base-methylated nucleosides was determined by continuous labeling of serum-stimulated quiescent hamster embryo cells with [3H-CH3]methionine and measurement of radioactivity in the excreted nucleosides between 23 and 81 1/2 hours after the label was added. These nucleosides accumulate exponentially until a maximum level is reached after 60 hours. These maximum levels were maintained for at least an additional 20 hours.

Salvage of the modified nucleoside ribothymidine in cultured hamster embryo cells.

RNA turnover in hamster embryo cells results in the release of ribothymidine, a methylated nucleoside formed only on intact RNA. [methyl-3H]Methionine ribothymidine polyphosphates and deoxyribothymidine polyphosphates are found in approximately equivalent amounts in the acid-soluble pool of hamster embryo cells. This observation is consistent with prior unexplained observations, in intact animals, that the methyl group of S-adenosyl methionine can be used for deoxythymidine synthesis, This is the only modified ribonucleoside known to be salvaged through pathways in normal cells. Several possible pathways for salvage were investigated.

Pyrimidine nucleotide pool changes during the cell cycle and quiescence. Pyrimidine excretion and metabolic isolation of the pyrimidine mononucleoside polyphosphate pool.

We have measured the pyrimidine nucleotide contents of the culture fluid, acid-soluble fraction, and acid-insoluble fraction of cultures of hamster embryo fibroblasts (third subculture) through the final two divisions of growth in culture. The cells show a growth delay between the penultimate and ultimate division periods and a concomitant biochemical synchrony of pyrimidine metabolism. The cells exhibit normal excretion of pyrimidine nucleotides beginning with the ultimate division cycle. This excretion results from the net breakdown of ribonucleic acid and a cell-regulated maximum for pyrimidine mononucleoside polyphosphate content. This upper limit for the pyrimidine nucleoside polyphosphate content is not a steady state phenomenon but rather an absence of both synthesis and utilization. The hamster embryo fibroblast exhibits a directed flow of salvage uridine for ribonucleic acid synthesis. We show that de novo synthetic uridine 5'-monophosphate also can be used for ribonucleic acid synthesis without prior entry into the cytoplasmic uridine nucleoside polyphosphate pool. During attachment and first division salvage uridine does enter the cytoplasmic nucleotide pool. The properties of the cytidine pools differ from the uridine pools in specific activity and levels of cytidine, due to turnover of the terminal C-C-A of cytoplasmic transfer ribonucleic acid and the delay in conversion of of nonradioactive de novo synthetic uridine 5'-monophosphate to cytidine 5'-triphosphate. The partial synchrony in these cultures has been used as a temporal marker of the observed events.

Rapid preparation of nucleotides from acid-soluble pools by chromatography on silica, as exemplified with acid extracts of cultured cells.

A rapid technique (5-10 min) has been developed for fractionating nucleotides from base and nucleoside contaminants in acid extracts of cells, by adsorption to silica gels. Silica gels (1-mL bed volume) were washed with 5 mL of water then with 5 mL of acetonitrile/water (90/10 by vol). After applying 3-mL samples, adjusted to 900 mL/L acetonitrile content, we washed the gel with an additional 10 mL of the acetonitrile/water solvent. More than 95% of the amounts of bases and nucleosides prsent, except for cytidine (92%), did not adsorb to silica under these conditions. Nucleotides were then quantitatively eluted with 9 mL of water. The retention volumes for positive, negative, and neutral nucleic acid components have been determined, to investigate the discriminatory properties of nucleic acid components on silica. Compounds (bases, nucleosides) that are not ionized at pH 7 do not bind to silica. However, negative, positive, and zwitterionic compounds are tightly adsorbed to the silica gels. This procedure has been used to purify nucleotides from several normal and transformed cell lines.

Pyrimidine nucleoside, pseudouridine, and modified nucleoside excretion by growing and resting fibroblasts.

We are examining the relationship of RNA metabolism and de novo pyrimidine synthesis as parameters of malignant transformation. These initial experiments on normal hamster embryo fibroblasts have shown that excreted nucleosides are markers for intracellular RNA metabolism. We employed affinity chromatography to concentrate the nucleosides in the medium and sensitive column chromatographic procedures to quantitatively measure them. The excretion of pyrimidine nucleoside from hamster embryo fibroblasts in sulture was found to be dependent on the growth state of the cells, with the greatest accumulation occurring cell quiescence. The major nucleoside excretion products, uridine and cytidine, were both normal end products of RNA metabolism and the major nucleoside excretion products from cultured cells. The modified nucleosides N-1-methylguanosine, N-2-methylguanosine, N-2-dimethylguanosine, N-4-acetylcytidine, N-1-methylinosine, pseudouridine, N-1-methyladenosine, N-3-methylcytidine, and 5-methyleycytidine were found, as were several unidentified nucleosides.

Pyrimidine biosynthesis in normal and transformed cells.

We have developed procedures for sensitive measurement of specific radioactivities of pyrimidine nucleosides excreted from cells in culture. The changes in the observed values reflect dilution of the added isotope through de novo biosynthesis of nonradioactive pyrimidine nucleosides or by shifting and equilibration of other nucleotide pools into the free uridine pool. It is thus possible to monitor uridine biosynthesis occurring in intact cells without destroying or disrupting the cell population. On comparing a series of normal and and transformed lines, we have observed several growth-dependent patterns of change in specific activity and levels of uridine excretion and the temporal appearance of these changes. Hamster embryo fibroblasts slows pyrimidine biosynthesis at mid-growth while the hamster cell line V79 continues to dilute the pyrimidine pool at about 7% of the rate observed during exponential growth at confluence. Both cells exhibit Urd excretion beginning at one-half maximal growth. Passageable normal rat liver cells (IARC-20) also show a cessation of pyrimidine biosynthesis with a prior increase in uridine excretion. Two chemically transformed lines IARC-28 and IARC-19 derived from IARC-20 show different patterns. IARC-19 begins uridine excretion in early log growth and the specific activity continues to decrease at about 2% of the rate observed during exponential growth at confluence. The IARC-28 cells also begin excretion in early log growth but pyrimidine biosynthesis stops at about midlog. This method may prove to be an additional aid in recognizing and differentiating transformed cells in culture that do not exhibit the transformed phenotype.

Modified nucleosides in urine: selective removal and analysis.

Because excretion of certain nucleosides increases in all types of cancer, detection and treatment of the disease would be enhanced by rapid, simple quantitation of such nucleotides. To facilitate these analyses in urine, we have prepared a specific adsorbent for nucleosides. The affinity adsorbent gel contains an immobilized phenylboronic acid group that specifically binds cis-diols, as in ribonucleosides, which can then be quantititatively recovered by elution with acetic acid. The advantage of this boronate gel over other preparations lies in its high capacity in the swollen state (0.26 mmol/ml), so that only small volumes (less than 1 ml) of gel are required for assays. Nucelosides containing a free diol group on the ribosyl group are retained at pH 8.8 in 0.25 mol/liter salt solution with a retention volume of 15 or larger, as compared with values of 1 to 2 for the free bases. Negatively charged species (not including the boronate complex) are eluted earlier than neutral or cationic species. Added nucleosides (neutral, cationic, or anionic) are quantitatively recovered. Because pseudouridine is eluted in a unique position, this property has been used to measure it in urine. The procedures used are chemically mild. For example, we have confirmed the observation of Fink and Adams [Arch. Biochem. Biophys. 126, 27 (1968)] that urine contains N1-methyladenosine and not N6-methyladenosine, the alkaline rearranged product.