A single column separation method for barium isotope analysis of geologic and hydrologic materials with complex matrices.

The increasing significance of barium (Ba) in environmental and geologic research in recent years has led to interest in the application of the Ba isotopic composition as a tracer for natural materials with complex matrices. Most Ba isotope measurement techniques require separation of Ba from the rest of sample prior to analysis. This paper presents a method using readily available materials and disposable columns that effectively separates Ba from a range of geologic and hydrologic materials, including carbonate minerals, silicate rocks, barite, river water, and fluids with high total dissolved solids and organic content such as oil and gas brines, rapidly and without need for an additional cleanup column. The technique involves off-the-shelf columns and cation exchange resin and a two-reagent elution that uses 2.5 N HCl followed by addition of 2.0 N HNO3. We present data to show that major matrix elements from almost any natural material are separated from Ba in a single column pass, and that the method also effectively reduces or eliminates isobaric interferences from lanthanum and cerium.

Multi-decadal geochemical evolution of drainage from underground coal mines in the Appalachian basin, USA.

Coal mine drainage (CMD) in Appalachia is a widespread source of dissolved metals, SO4, and acidity that can degrade aquatic habitats and water supplies for decades following mine closure and flooding. In the bituminous coalfield of Pennsylvania, the Irwin Coal Basin (ICB) contains a series of partly to completely flooded, abandoned underground mines separated by leaky barriers within the Pittsburgh coal seam. CMD originated throughout the basin from minepool aquifers that formed after mine closures dating from 1910 to 1957. Historical and recent water quality data for eight CMD sites across the ICB, plus mineralogy and cation-exchange capacity of overburden lithologies, were analyzed to quantify important reactants and evaluate spatial and temporal water-quality trends. As overburden thickness and residence time increase along a ~ 50-km flowpath northeast to southwest in the basin, CMD becomes more alkaline, and Na concentrations increase. Since the 1970s, all eight ICB discharges have become less acidic, with exponential decreases in acidity, SO4, and Fe concentrations; only two CMD remain net-acidic (acidic pH at equilibrium). Exponential decay models that include a steady-state asymptote consistent with background groundwater chemistry and siderite equilibrium describe the early-stage, rapid contaminant concentration decay immediately after the "first flush" (initial flooding) and the progressive evolution toward late-stage background conditions. A geochemical evolution PHREEQC model indicates that spatial and temporal trends in pH, net-acidity, SO4, Fe, and major cations could be explained by the continuous dilution of first flush water by ambient groundwater combined with sustained water-mineral reactions involving pyrite and carbonates (calcite, dolomite, siderite) plus cation-exchange by clays (illite, chlorite, mixed-layer illite/smectite). These data and model results indicate that 1) cation-exchange reactions enhance calcite dissolution and alkalinity production, resulting in the evolution of CMD to Na-SO4-HCO3 type waters, and 2) siderite equilibrium could maintain dissolved Fe >16 mg/L over the next 40 years.

Diffusive isotopic contamination of mafic magma by coexisting silicic liquid in the muskox intrusion.

Shifts in (87)Sr/(86)Sr and (143)Nd/(144)Nd ratios measured in cumulates from the upper levels of the Muskox mafic intrusion indicate that isotopic and bulk chemical exchange were decoupled across a mafic-silicic liquid interface during crystallization of the intrusion. Modeling of diffusive exchange between liquid layers demonstrates that isotopic compositions of silicate liquids in layered magma chambers may be strongly affected by this process on time scales of 10(3) to 10(4) years. Diffusive contamination can be used to place constraints on the physical processes and time scales of magmatic systems.

Mechanisms of apoptosis: integration of genetic, biochemical, and cellular indicators.

Apoptosis, or programmed cell death, is defined by morphologic change resulting in nonpathologic cell loss and is relevant to a wide spectrum of biology. The process is best characterized in the nematode Caenorhabditis elegans where ced genes mediate the death of specific cells during development. Some corresponding genes have been identified in mammalian cells. Expression of the mammalian bcl-2 gene (homologous to ced-9) suppresses apoptosis in many systems. The ced-3 gene is homologous to a mammalian protease. Increased levels of the tumor suppressor p53 due to DNA damage may result in either blockage of the cell cycle at G1 or apoptosis. Mutation of p53 is associated with decreased cell death from radiation and cytotoxic drugs. Initiation of the apoptotic pathway may occur as a consequence of conflicting growth signals. Hierarchical relationships variously between bcl-2, p53, myc, and other genes indicate a complex pattern of regulation. Stimuli resulting in apoptosis may cause production of free radicals and increased intracellular calcium concentration. The relationship of these changes to the hallmark of apoptosis, internucleosomal fragmentation of DNA, is unclear, and "laddering" of DNA is not always evident. Apoptotic DNA degradation probably occurs sequentially, initially involving breakage into 50 kilobases or larger fragments. The nuclease(s) responsible have not been identified, but deoxyribonuclease I is implicated. The association between nuclease activation and chromatin condensation is complex, and programmed cell death may be subject to cytoplasmic regulation. Available data suggest that clearer understanding of apoptosis will result in better cancer therapy.

Resistance to drugs associated with the multidrug resistance phenotype following selection with high-concentration methotrexate.

To study patterns of resistance at extreme but nevertheless clinically relevant drug concentrations, we developed a series of methotrexate-selected CCRF-CEM sublines, all of which were highly resistant to this antifolate (relative resistance, 10(2)- to greater than 10(5)-fold). The least methotrexate-resistant subline was completely sensitive to drugs associated with the multidrug resistance phenotype. However, more highly methotrexate-resistant sublines were significantly cross-resistant to vincristine, vinblastine, and dactinomycin (maximum relative resistance, 40-fold). These sublines were not cross-resistant to doxorubicin, daunorubicin, and teniposide. Regression analysis indicated that relative resistance to methotrexate was correlated with relative resistance to vincristine (r = 0.96) and vinblastine (r = 0.99). Such cross-resistance in highly methotrexate-resistant cells may have important clinical implications.

Generation and persistence of carcinogen-induced repair intermediates in rat liver DNA in vivo.

Chromatographic separation of native DNA from DNA containing single-stranded regions has been used to determine the relative concentrations of structural intermediates generated during chemically induced DNA repair. Single doses of each of ten compounds were administered to rats. After periods ranging from 90 min to 13 days, hepatic DNA was isolated and analyzed by stepwise elution from benzoylated diethylaminoethyl cellulose with 1.0 M NaCl followed by caffeine solution. The compounds used were benzo(a)pyrene, carbon tetrachloride, diethylnitrosamine, dimethylnitrosamine, ethyl methanesulfonate, galactosamine, N-hydroxy-2-acetylaminofluorene, methyl methanesulfonate, nitrosomorpholine, and beta-propiolactone. Doses of the various agents and/or treatment times were restricted such that hepatic necrosis did not occur. No increase in the amount of caffeine-eluted DNA occurred after administration of carbon tetrachloride or galactosamine. All the remaining chemicals caused a dose-dependent increase in the proportion of hepatic DNA eluted from benzoylated diethylaminoethyl cellulose with caffeine. In most cases, the varying times required to produce maximal increase in the proportion of caffeine-eluted DNA could be related to the rate of metabolism of the carcinogens. A distinction could be made according to whether repair intermediates were detected only within 24 hr of administration (ethyl methanesulfonate, methyl methanesulfonate, and beta-propiolactone) or were present for at least 3 days after treatment (diethylnitrosamine, dimethylnitrosamine, benzo(a)pyrene, N-hydroxy-2-acetylaminofluorene, and nitrosomorpholine). The data, considered with reference to previously ascribed modes of DNA repair for the respective adducts, suggest that base excision repair is immediately operative and rapidly completed in rat liver. However, reactions involved in the completion of nucleotide excision repair may be rate limiting, resulting in persistent structural damage to DNA. Implications of these findings for the use of benzoylated diethylaminoethyl cellulose chromatography as a carcinogen bioassay are considered.

Etoposide-induced cytotoxicity in two human T-cell leukemic lines: delayed loss of membrane permeability rather than DNA fragmentation as an indicator of programmed cell death.

Features of the apoptotic response evident in glucocorticoid-treated thymocytes are not uniformly observed in cell lines exposed to anticancer drugs. The significance of such variation has been assessed by monitoring molecular and cellular processes induced by etoposide (VP-16) in the human lymphoblastoid T-cell lines CCRF-CEM (CEM) and MOLT-4 contrasted, where appropriate, with those induced by necrotizing injury. Cytotoxic concentrations of the drug were determined to be 5-100 microM on the basis of tetrazolium reduction assay. The two lines were equally sensitive to VP-16; no difference in concentration of drug which inhibited cell growth by 50% with respect to control (i.e., drug free) cultures was apparent irrespective of exposure times from 3-72 h. DNA strand breaks were evident in both populations within 3 h of exposure to VP-16. Morphological change, assessed microscopically, involving nuclear condensation and cell shrinkage was qualitatively and quantitatively similar in VP-16-treated CEM and MOLT-4 cells. Flow cytometric analysis indicated that the G2/M fraction of the randomly dividing MOLT-4 population was approximately one-third that of CEM cells, but each line exhibited a decrease in this fraction 3-6 h after treatment. Despite these similarities, marked differences in the response to VP-16 were evident in the two populations. Internucleosomal fragmentation, detected electrophoretically 3 h after treatment in DNA isolated from CEM cells, was not detected under any condition in MOLT-4 DNA. Apoptotic bodies, also evident within 3 h of VP-16 treatment of CEM cells, were not readily apparent in MOLT-4 cells under the same conditions. Treatment causing necrosis resulted in trypan blue uptake within 1 h in a similar high proportion of cells from both lines. Exposure to VP-16 resulted in such a loss of membrane integrity by 6 h in CEM cells, while change in this parameter occurred only after 24 h in the case of MOLT-4 cells. The findings indicate a wide scope of difference in apoptotic response and suggest delayed loss of membrane permeability, rather than DNA fragmentation, as the clearest indicator of programmed cell death in these populations.

Alterations in thermal stability of rat liver chromatin and DNA induced in vivo by dimethylnitrosamine and diethylnitrosamine.

A study was made of the effects of administration to rats of dimethylnitrosamine and diethylnitrosamine on the transition temperature (Tm) of sheared chromatin and DNA isolated from the liver. The analysis was made by thermal chromatography on hydroxylapatite with the use of DNA prelabeled with [3H]thymidine and following the elution pattern during the operation of a continuous temperature gradient. With a nonnecrogenic dose of dimethylnitrosamine (10 mg/kg), the alterations in chromatin were maximal at 24 hr and disappeared by 3 days. Greatest differences in elution profiles of chromatin after dimethylnitrosamine treatment were observed in the region above 80 degrees. Administration of the carcinogen caused a lowering of the "melting" curve in this region, the displacement from control position being proportional to the dose. The maximum dose (60 mg/kg) displaced the complete chromatin melting curve up to 5 degrees to the lower side. DNA isolated from this chromatin melted 3 degrees less than that from control rats. However, administration of lower doses of dimethylnitrosamine did not affect the melting profile of DNA. The administration of diethylnitrosamine caused a similar type of change. However, the modification was also seen at 50-60 degrees.

Heterotransplantation of human lymphoid neoplasms using a nude mouse intraocular xenograft model.

There are few effective models for the study of human lymphoid neoplasms, including in vivo xenografts in immunocompromised animals. Exploiting the additional immune privilege of the anterior chamber of the nude mouse eye, a novel method of direct heterotransplantation of cells from childhood leukemias and lymphomas has been developed. The establishment and characterization of 18 lymphoid xenograft cell lines maintained in the nude mouse intraocular model are reported. Cell sources for heterotransplantation were specimens of bone marrow, peripheral blood, or lymphomatous masses obtained at either diagnosis or recurrence of disease in the patients. The 18 patients and resultant cell lines were grouped into four immunophenotypic categories: Category 1, B-lineage (pre-B and early pre-B), "common" acute lymphatic leukemias; Category 2, cell lines of similar immunophenotype derived from patients with unusual features; Category 3, B-cell neoplasms and cell lines; and Category 4, neoplasms and cell lines in part or totally of T-cell origin. With reference to these groupings, rates of ingraftment from clinical specimens varied according to immunophenotype and disease status: Category 1, 1 of 15 at diagnosis, 5 of 7 at relapse; Category 2, 1 of 1 at diagnosis, 2 of 2 at relapse; Category 3, 6 of 6 at diagnosis; and Category 4, 2 of 9 at diagnosis, 1 of 1 with persistent disease. Rearrangements of the genes for immunoglobulin heavy chain or kappa light chain and for beta subunit of the T-cell receptor gene were demonstrated according to immunophenotype, with the exception of one cell line which showed no rearrangements. Evidence of Epstein-Barr virus DNA was shown in only one cell line, of B-cell immunophenotype. Cytology, histopathology, and electron microscopy in representative patient and xenograft samples demonstrated correlations between the specimens of origin and cells or sections from ingrafted tumors in mice. It is concluded that the direct heterotransplantation of cells from childhood leukemias and lymphomas to the anterior chamber of the nude mouse eye provides a relevant and reproducible model for the maintenance and study of human lymphoid neoplasms.

Atypical multidrug resistance in a therapy-induced drug-resistant human leukemia cell line (LALW-2): resistance to Vinca alkaloids independent of P-glycoprotein.

Near diploid leukemic T-cells (LALW-2), exposed to cytotoxic drugs only as a consequence of therapy administered to the donor patient, have been maintained by serial xenograft in nude mice. In comparison with the leukemic line CCRF-CEM, using a growth inhibition assay, LALW-2 cells were resistant to Vinca alkaloids and actinomycin D (relative resistance, 200-fold or more), were slightly resistant to Adriamycin (relative resistance, 4-fold), and showed no resistance to daunorubicin or teniposide. By comparison, a vincristine-resistant CEM subline developed in our laboratory (CEM/VCR R) was resistant to all these agents by at least 30-fold. The VCR R subline served as a positive control, confirming the previously reported correlation between multidrug resistance and amplification of the P-glycoprotein gene. Comparison of CEM, CEM/VCR R, and LALW-2 cells establish that the P-glycoprotein gene was not amplified or overexpressed in the LALW-2 cells; neither could the gene product be detected by immunoblotting in extracts from these cells. The LALW-2 cells were further distinguished from CEM/VCR R cells due to the lack of increased vincristine efflux by the xenografted cells, an effect readily demonstrable in the CEM/VCR R cells. However, although LALW-2 cells efflux vincristine at the same rate as CCRF-CEM cells, the xenografted cells exhibited a reduced rate of vincristine accumulation. Uptake of daunorubicin by LALW-2 cells was not distinguished from that by CEM cells, consistent with similar 50% inhibitory dose levels for this drug in both cell populations, and differentiating both from CEM/VCR R cells. Thus, clinical resistance in this case appears to be an "atypical" form of multidrug resistance specifically distinguished by resistance to Vinca alkaloids and actinomycin D occurring in the absence of increased amounts of P-glycoprotein and manifesting decreased drug uptake.

Morphological and molecular evidence of differentiation during etoposide-induced apoptosis in human lymphoblastoid cells.

The relationship between apoptosis and cell differentiation has been a subject for continuous debate, with evidence showing leukaemic cell differentiation and drug-induced apoptosis have reciprocal, interdependent and a highly schedule-dependent relationship. We have addressed this relationship in terms of a widely-used model for apoptosis induced by cytotoxic drugs: namely the effect of etoposide on CEM cells. In respect of commitment toward differentiation, we assessed changes in expression of marker genes and the level of CD3 antigenicity. Changes in these parameters following exposure of CEM cells to etoposide was similar to that observed following treatment of the same cells with phorbol 12-myristate 13-acetate (PMA), though this latter treatment did not cause cell death. Similarities in response to etoposide and PMA also included generation of 50 kilobase fragmentation of DNA and convolution of nuclei as assessed by transmission electron microscopy. However, condensation of chromatin and externalization of phosphatidylserine were only recorded in response to the cytotoxic drug and not in response to PMA. The data are consistent with apoptosis in these lymphoblastoid cells being accompanied by activation of specific markers of T-cell differentiation, but ultimately involving processes unequivocally associated with cell death.

Early caspase activation in leukemic cells subject to etoposide-induced G2-M arrest: evidence of commitment to apoptosis rather than mitotic cell death.

After exposure to cytotoxic drugs at relatively low concentration, many cell types undergo G2-M arrest and then either mitotic cell death or, in the case of hematopoietic cells, apoptosis. We have sought to examine this phenomenon in two lymphoblastoid cell lines. After continuous or short-term exposure to etoposide (final concentration, 0.5 microM), up to 80% of cells accumulated at G2-M by 24 h, and subsequently either underwent apoptosis or re-entered the cell cycle. In this and the other studies undertaken, the CEM and MOLT-4 lines behaved similarly. Progressive accumulation of cells at G2-M was accompanied by increasing levels of cyclin B1. Commitment to apoptosis was assessed by evidence of caspase activation using a number of different criteria. A decreased amount of Mr 32,000 procaspase-3 was evident 24-48 h after drug treatment. However, cleavage of caspase substrates poly(ADP-ribose) polymerase and lamin B indicated caspase activation occurring within 3-6 h of drug treatment. Protease activity in corresponding cell extracts increased progressively from 6 h or earlier to 24 h after the addition of etoposide to the medium. Such increase was consequent on drug treatment and not attributable to cells being at G2-M. Treatment with 1.5 mM caffeine abrogated etoposide-induced G2-M arrest, and in cells so treated, the etoposide-induced increase in protease activity was also abrogated. However, there was no impact of caffeine on cytotoxicity under these conditions. Although mitotic cell death is precipitated subsequent to prolonged G2-M arrest in many cell types, the present data suggest that commitment to apoptosis occurs in parallel to G2-M arrest in leukemic cells.

Sizing of single-stranded regions in double-stranded DNA by preparative benzoylated DEAE-cellulose chromatography.

The concentration of caffeine required to elute wholly single-stranded DNA from benzoylated DEAE-cellulose is proportional to the polynucleotide length. The use of benzoylated DEAE-cellulose chromatography for isolating and sizing single-stranded regions in double-stranded DNA has been examined using a series of hybrid molecules. Restriction fragments of the replicating form of bacteriophage luminal diameter X174 were hybridized to the intact 'plus' strand, thereby forming hybrids having single- and/or double-stranded regions in the kilobase range. A series of such hybrid preparations were subject to caffeine concentration gradient elution from benzoylated DEAE-cellulose. After logarithmic transformation, a linear relationship (R = 0.94) could be demonstrated between eluting caffeine concentration and single-stranded length, irrespective of the length of associated double-stranded regions or the location, within a given fragment, of unpaired nucleotides. Benzoylated DEAE-cellulose chromatography may therefore be used to separate and characterize, on a preparative scale, double-stranded DNA containing single-stranded regions.

DNA-mediated gene transfer as an indicator of DNA damage and its repair by recipient cells.

Preparations of plasmid containing the thymidine kinase gene (pHSV106) were treated with the alkylating agents methyl methanesulphonate or N-methyl-N-nitrosourea prior to transfection into thymidine kinase-deficient mouse L-cells using the DNA-calcium phosphate co-precipitation technique. Relative to transfection with unmodified plasmid, a reduced transformation efficiency was observed using alkylation-damaged plasmid, N-methyl-N-nitrosourea causing the greatest inhibition. Treatment of recipient cells with arabinosyl cytosine or dideoxythymidine during the expression period following transfection by the 'damaged' plasmid reduced transformation efficiency, suggesting that DNA repair 4-6 h post-transfection was required for gene expression.

The prognostic value of MDR1 gene expression in primary untreated neuroblastoma.

The contribution of MDR1 gene expression to the biology of childhood neuroblastoma is unclear. To clarify the role of MDR1 in this malignancy, we examined the relationship between MDR1 expression and patient outcome in subsets of 60 primary untreated neuroblastomas for which MYCN gene copy number and expression of the multidrug resistance-associated-protein (MRP) gene had been previously characterised. In contrast to MRP gene expression, MDR1 expression was lower in tumours with MYCN gene amplification compared with those without amplification. Strong correlations between MDR1 and MRP gene expression, and between MDR1 and MYCN gene expression, were observed in tumours lacking MYCN gene amplification (P < 0.0005). In these single-copy tumours, very high MDR1 gene expression was significantly associated with poor outcome (P < 0.05). Very high MDR1 expression was also strongly predictive of poor outcome in older children (P < 0.0001), but not in infants. These findings suggest a clinical role for the MDR1 gene in specific subgroups of primary neuroblastoma.

Dimethylnitrosamine-induced structural damage to DNA results from repair of O6-methylguanine rather than repair of 7-methylguanine.

DNA isolated from the livers of rats treated with a non-necrotizing dose of [14C]dimethylnitrosamine was fractionated by chromatography of benzoylated-DEAE-cellulose. The preparations of native DNA and DNA containing single stranded regions were then hydrolysed and amounts of methylated guanine determined. Four hours after dimethylnitrosamine treatment there was no difference in the levels of 7-methylguanine and O6-methylguanine in the 2 DNA fractions. By 24 h, although there was no difference in the amount of 7-methylguanine between the DNA fractions, there was a 10-fold difference in the level of O6-methylguanine. The elimination of O6-methylguanine from the fraction of DNA containing single stranded regions is discussed in terms of differing repair processes initiated by 7-methylguanine and O6-methylguanine.

Repair and de novo replication of rat lung DNA following instillation of 3-methylcholanthrene.

Structural analysis of rat lung DNA was made by stepwise elution of the nucleic acid from benzoylated--DEAE-cellulose with 1.0 M NaCl and 30% formamide solutions respectively, the DNA content of collected fractions being determined by absorbance. The proportion of the minor fraction, which was eluted with formamide and thought to contain DNA having single-stranded regions, was increased by intratracheal instillation of 3-methylcholanthrene prior to death of the animal. Maximum initial increase was observed 14 h after treatment. Analysed up to 10 days after treatment, there was a biphasic response, the early maxima being dose dependent and the second apparently independent of the amount of hydrocarbon administered. The data implicate DNA repair processes and cellular proliferation stimulated by 3-methylcholanthrene as being involved in recovery of the lung from this potentially carcinogenic insult.

N-(4-hydroxyphenyl)retinamide-induced death in human lymphoblastoid cells: 50 kb DNA breakage as a means of distinguishing apoptosis from necrosis.

Experimental studies of N-(4-hydroxyphenyl)retinamide, a potential cancer chemopreventive agent, have primarily involved breast cancer and neuroblastoma cell populations together with an investigation of myeloid leukemia cells and have principally been concerned with the induction of apoptosis. This investigation of N-(4-hydroxyphenyl)retinamide-induced apoptosis using T-cell-derived human lymphoblastoid lines extends these studies by indicating distinctive features associated with this drug. The induction of apoptosis is restricted to a limited concentration range, which, if exceeded, results in cell death by necrosis. While morphological changes typical of apoptosis induced by many agents are readily demonstrable after treatment of lymphoblastoid cells with 3 microM N-(4-hydroxyphenyl)retinamide, distinctive features evident using the retinoid include the absence of cell cycle arrest along with the mode and pattern of DNA breakage. Analysis by conventional gel electrophoresis indicated that internucleosomal fragmentation of DNA was an unreliable indicator of apoptosis. On the other hand, higher order DNA breakage was consistently detected during drug-induced apoptosis, but not as a result of treatment causing necrosis.

Transient structural change in hepatic DNA of rats chronically exposed to dimethylnitrosamine.

Structural analysis by chromatography on benzoylated-DEAE-cellulose (BD-cellulose) has been made of hepatic DNA from rats treated for up to 21 weeks with dimethylnitrosamine (DMN). The carcinogen (1 mg/kg body wt) was administered on a daily basis by intraperitoneal injection. By comparison with preparations from saline-treated controls, the proportion of DNA retained by benzoylated-DEAE-cellulose in the presence of 1.0 M NaCl was increased by administration of the carcinogen. Variation in the result, dependent upon the time after the final dose, suggested a complex relationship between structural damage to DNA and duration of treatment. Structural damage was confirmed by S1 nuclease digestion. The observations imply that in the course of chronic administration, increasing time is required to complete DNA repair processes operative in rat liver.