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.

Designated "no smoking" areas provide from partial to no protection from environmental tobacco smoke.

OBJECTIVE: To determine the efficacy of designated "no smoking" areas in the hospitality industry as a means of providing protection from environmental tobacco smoke (ETS), and whether certain design features assist in achieving this end. METHODOLOGY: In the greater metropolitan region of Sydney, a representative group of 17 social and gaming clubs, licensed to serve alcoholic beverages and in which, apart from designated areas, smoking occurs, agreed to participate. In each establishment, simultaneous single measurements of atmospheric nicotine, particulate matter (10 microm; PM10) and carbon dioxide (CO2) levels were measured in a general use area and in a designated "no smoking" area during times of normal operation, together with the levels in outdoor air (PM10 and CO2 only). Analyses were made of these data to assess the extent to which persons using the "no smoking" areas were protected from exposure to ETS. RESULTS: By comparison with levels in general use areas, nicotine and particulate matter levels were significantly less in the "no smoking" areas, but were still readily detectable at higher than ambient levels. For nicotine, mean (SD) levels were 100.5 (45.3) microg/m3 in the areas where smoking occurred and 41.3 (16.1) microg/m3 in the "no smoking" areas. Corresponding PM10 levels were 460 (196) microg/m3 and 210 (210) microg/m3, while outdoor levels were 61 (23) microg/m3. The reduction in pollutants achieved through a separate room being designated "no smoking" was only marginally better than the reduction achieved when a "no smoking" area was contiguous with a smoking area. CO2 levels were relatively uninformative. CONCLUSION: Provision of designated "no smoking" areas in licensed (gaming) clubs in New South Wales, Australia, provides, at best, partial protection from ETS-typically about a 50% reduction in exposure. The protection afforded is less than users might reasonably have understood and is not comparable with protection afforded by prohibiting smoking on the premises.

Trichloroethylene and cancer: a carcinogen on trial.

The organic solvent trichloroethylene has been used in dry cleaning, as an industrial degreasing agent and as a solvent for oils and resins; large numbers of workers have been exposed to trichloroethylene, mainly by inhalation. Trichloroethylene has been categorised as a Group 2A carcinogen (probably carcinogenic to humans) by the International Agency for Research on Cancer (World Health Organization) and a Category 2 carcinogen (to be regarded as carcinogenic to humans) by the Australian National Industrial Chemicals Notification and Assessment Scheme. The Administrative Appeals Tribunal was asked to determine the validity of classifying trichloroethylene as a Category 2 rather than a Category 3 (data inadequate for making a satisfactory assessment) carcinogen. In the AAT's determination, relevant epidemiological evidence was not taken into account because such evidence concerned tumour sites apart from the kidney (the site of tumour induction by trichloroethylene in rats). This mode of evaluation is fundamentally different from that used by the International Agency for Research on Cancer. The precedent set by the consideration of carcinogenicity data in this case could have significant implications for classification of other putative carcinogens

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.

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.

Investigation of a cluster of leukaemia in the Illawarra region of New South Wales, 1989-1996.

OBJECTIVES: To investigate a cluster of leukaemia among young people and assess the plausibility of a disease-exposure relationship. DESIGN: Descriptive analysis of population-based leukaemia incidence data, review of evidence related to the causation of leukaemia, assessment of environmental exposures to known leukaemogens, and resulting risks of leukaemia. SETTING: Illawarra region of New South Wales, Australia, focusing on suburbs between the Port Kembla industrial complex and Lake Illawarra (the Warrawong area). MAIN OUTCOME MEASURES: Standardised incidence ratios (SIRs) for leukaemia; current measured and past estimated ambient air benzene concentrations; and expected leukaemia cases attributable to estimates of ambient air benzene concentrations. RESULTS: In 1989-1996, 12 leukaemia cases among Warrawong residents aged less than 50 years were observed, more than the 3.49 cases expected from the rate in the rest of the Illawarra region (SIR, 343.8; 99% CI, 141.6-691.7). These people lived in suburbs immediately to the south-southwest of a coke byproducts plant (a major industrial source of benzene, one of the few known leukaemogens). The greatest excess was among 15-24-year-olds (SIR, 1085.6; 99% CI, 234.1-3072.4). In 1996, ambient air concentrations of benzene averaged less than 1 part per billion (ppb). Since 1970, ambient air concentrations of benzene were estimated to have averaged up to 3 ppb, about one-thousandth of the level at which leukaemia risk has been identified in occupational epidemiological studies. Using the risk assessment model developed by the US Environmental Protection Agency, we estimate that past benzene levels in the Warrawong area could have resulted in 0.4 additional cases of leukaemia in 1989-1996. CONCLUSIONS: The excess occurrence of leukaemia in the Warrawong area in 1989-1996 is highly unusual. Current environmental benzene exposure and the reconstructed past environmental benzene exposure level are too low to explain the large excess of leukaemia. The cause of the cluster is uncertain.

P-glycoprotein-mediated methotrexate resistance in CCRF-CEM sublines deficient in methotrexate accumulation due to a point mutation in the reduced folate carrier gene.

We have previously described a series of methotrexate (MTX)-selected CCRF-CEM sublines (CEM/MTX R1-3) displaying increased resistance to drugs associated with the multidrug resistance phenotype and have provided evidence that MDR1 P-glycoprotein contributes to multifactorial MTX resistance in these cells. We have also suggested that P-glycoprotein-mediated MTX transport arises in these cells due to a deficiency in the normal MTX transport route, the reduced folate carrier (RFC). We have now determined the nucleotide sequence of the RFC gene in CEM/MTX R1-3 cells and confirm that the carrier is defective in these cells as a result of a premature stop mutation at codon 99, which severely truncates the encoded protein. CEM/MTX R3 cells were removed from MTX, and a series of sublines with increasing MDR1 expression were derived, following selection with vincristine. These cells show increasing cross-resistance to vincristine as well as other drugs associated with the multidrug resistance phenotype. More importantly, the increased P-glycoprotein expression correlates with increased resistance to MTX, supporting the hypothesis that in cells with a defective carrier protein, MTX can become a substrate for P-glycoprotein. Our data have implications for the P-glycoprotein-mediated transport of other hydrophilic drugs in situations where the relevant carrier protein has been functionally inhibited.

Growth inhibition of HL-60 cells by extracellular ATP: concentration-dependent involvement of a P2 receptor and adenosine generation.

A single addition of ATP (20-1000 microM) to cultures of HL-60 cells resulted here in permanent, Ca(2+)-independent inhibition of cellular proliferation, evident 48 h following treatment. Extracellular ATP (ATPo) was maximally effective at 250 microM giving 90 +/- 1.5% growth inhibition. Up to a concentration of 250 microM ATPo, growth inhibition is solely attributable to ATPo, while at higher ATPo concentrations adenosine generated from ATPo hydrolysis contributes to this effect. The order of potency for growth inhibition was ATP = ADP > AMP > adenosine. Suramin, a P2 receptor antagonist, attenuated growth inhibition by ATP and ADP, indicative of P2 receptor involvement. Equipotency of ATP and ADP excludes the involvement of either an ecto-protein kinase or a P2X7 receptor in growth inhibition. Neither UTP (P2Y2 agonist) nor alpha, beta-methyleneATP (P2X1 agonist) inhibited growth, indicating that such inhibition is mediated by a previously undescribed P2 receptor on HL-60 cells.

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.

Drug-induced death of leukaemic cells after G2/M arrest: higher order DNA fragmentation as an indicator of mechanism.

Many reports have documented apoptotic death in different cell types within hours of exposure to cytotoxic drugs; lower drug concentrations may cause cell cycle arrest at G2/M and subsequent death, which has been distinguished from 'classic' apoptosis. We have analysed etoposide-induced cell death in two lymphoblastoid T-cell lines, CCRF-CEM and MOLT-4, specifically in relation to DNA cleavage as indicated by pulse-field gel and conventional electrophoresis. High (5 microM) concentration etoposide causes 50-kb cleavage of DNA that occurs at the same time as apoptotic morphology and internucleosomal cleavage. At lower concentrations (0.5-0.05 microM), sequential change may be discerned with altered gene expression being similar to that at high dose, but preceding cell cycle arrest and 50-kb cleavage. These last changes, in turn, clearly precede internucleosomal fragmentation of DNA, vital dye staining and morphological evidence cell death. The pattern of higher order fragmentation constitutes a sensitive indicator of commitment to cell death in these cells. Morphological evidence of cell death is associated with internucleosomal fragmentation in one of the lines, but the pattern of 50-kb DNA cleavage provides the clearest evidence of commonality in death processes occurring at low and high drug concentration.

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.

Retinoic acid receptors beta and gamma distinguish retinoid signals for growth inhibition and neuritogenesis in human neuroblastoma cells.

Retinoids induce marked growth inhibition and neuritic differentiation in human neuroblastoma cells. Expression patterns of nuclear retinoic acid receptors (RAR) in embryonic and adult tissues suggests that RAR subtypes alpha, beta and gamma have tissue-specific functions. We have transfected a human neuroblastoma tumor cell line with a vector expressing either human RAR alpha, beta or gamma cDNAs. In the absence of exogenous retinoid, RAR beta transfectants demonstrated marked growth inhibition without morphologic evidence of differentiation, whereas transfectant clones overexpressing RARs alpha and gamma had no significant reduction in cell growth rates. Although RAR gamma transfectants were sensitive to the growth inhibitory effects of exogenous retinoids, these cells demonstrated resistance to the neuritogenic retinoid effects. Only RAR beta transfectants exhibited increased sensitivity to retinoids added in vitro. These results suggest that distinct neuritogenic and growth inhibitory signalling pathways exist in neuroblastoma cells and that RAR beta expression may be necessary for the retinoid growth inhibitory pathway.

Involvement of MDR1 P-glycoprotein in multifactorial resistance to methotrexate.

Cellular resistance to methotrexate (MTX) is believed to be unaffected by expression of MDR1 P-glycoprotein (Pgp), a pleiotropic efflux pump acting on different hydrophobic compounds that enter cells by passive diffusion. A series of human leukemic CCRF-CEM sublines, isolated by multi-step selection for very high resistance to MTX, exhibit multiple mechanisms of MTX resistance, including decreased carrier-mediated uptake of MTX and DHFR gene amplification. These sublines show cross-resistance to drugs of the multi-drug resistance (MDR) family, which is correlated with relative resistance to MTX. The MTX-selected sublines show increased expression and function of the MDR1 gene, based on the measurement of MDR1 mRNA, Pgp and rhodamine 123 accumulation. Sequence analysis of the MDR1 cDNA from MTX-selected CCRF-CEM cells revealed no mutations in the protein coding region. MTX resistance in these cell lines is partially reversible by a Pgp-specific monoclonal antibody (MAb) UIC2 and a monovalent FaB fragment of UIC2. Our results indicate that Pgp can contribute to multifactorial resistance to MTX.

Data evaluation as a contribution to cancer prevention.

Current evidence of chemopreventive activity for a variety of agents, from epidemiological studies and/or through laboratory investigations, is adequate to justify a range of clinical trials. These circumstances have prompted the International Agency for Research on Cancer to consider an International data evaluation programme in cancer prevention, analogous to the Monographs programme for carcinogens. Aspects of the Monographs programme that have contributed to its success merit examination in this context. It is clear that any new data evaluation programme must be grounded in a multidisciplinary assessment of data: relevant expertise extends from molecular genetics through to the design and evaluation of clinical trials. At the end, preventive efficacy for whole populations is unlikely: evaluations will probably indicate efficacy of an agent in relation to a particular organ or tumour type and in high-risk groups. Virtually all putative chemopreventive agents have adverse effects; many are carcinogenic. Such indications of hazard impinge upon the possible usage of these same agents to prevent cancer. Quantitative data concerning dose-specific effects, where available, will be relevant. It will also be important to elucidate the biological processes that account for chemoprevention and carcinogenesis - and to consider mechanisms relevant to other biological end points. Publication of evaluations of chemopreventive data may cause difficulty if such evaluations are misused or quoted out of context. This consideration and other complexities associated with the wider use of chemopreventive agents are discussed.

An assay for the determination of reduced methotrexate accumulation in cells displaying limited viability in vitro.

Amongst the mechanisms known to mediate resistance to methotrexate (MTX), a major component in the treatment of childhood leukemia, reduced drug accumulation appears to have direct clinical relevance. However, due to the poor viability of patient-derived acute lymphoblastic leukemia cells in vitro, determination of this parameter in clinical samples is associated with a number of difficulties. We have therefore developed an assay for reduced MTX accumulation, which controls for the metabolic state of the cell population under study by utilizing accumulation of the nucleoside thymidine as an independent indicator of this parameter. To establish this assay, we have utilized pediatric leukemic cell populations maintained as xenografts in nude mice, which, despite displaying sensitivity to MTX, demonstrated reduced accumulation of MTX when assayed using standard methodology. When accumulation of MTX by such cell populations was expressed, however, relative to their accumulation of thymidine, MTX accumulation was shown to be equal to that of drug-sensitive CCRF-CEM cells maintained in long-term culture. In contrast, significantly less MTX was accumulated, in this assay, by xenografted cell populations with demonstrated resistance to MTX. Identical results were obtained using either fresh or cryopreserved cells. The data thus indicate that by controlling for variable metabolic status of leukemic cells, it is possible to accurately assess MTX accumulation in leukemic samples displaying limited viability in culture.

Formation of apoptotic bodies is associated with internucleosomal DNA fragmentation during drug-induced apoptosis.

The onset of apoptosis is often coincident with internucleosomal DNA fragmentation or ladders which are considered a hallmark of the process. However, several studies have indicated that MOLT-4 human lymphoblastoid cells exposed to various agents, including VP16, display some apoptotic characteristics in the absence of either internucleosomal ladders or production of apoptotic bodies. The present study records that, in the presence of aurintricarboxylic acid (ATA), internucleosomal ladders were detected in DNA isolated from VP16-treated MOLT-4 cells; a paradoxical result in view of inhibition by ATA of nuclease activity in cell free preparations. The activity of ATA in mediating genomic fragmentation was dose- and time-dependent. Moreover, addition of ATA to VP16-treated MOLT-4 cells also resulted in production of apoptotic bodies, this effect being quantified by morphological examination and flow cytometry. Detection of ladders and apoptotic bodies after addition of ATA was not attributable to increased toxicity in cells exposed to the combined treatment relative to VP16 alone. A similar response, that is the appearance of both internucleosomal fragmentation and apoptotic bodies, occurred after exposure of MOLT-4 cells to the mitotic inhibitor podophyllotoxin. The consistent association between internucleosomal fragmentation of DNA and formation of apoptotic bodies exhibited during death of MOLT-4 cells, insofar as both characteristics are either present or absent following different agents, suggests interdependence.

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.

Inhibition of topoisomerase II by aurintricarboxylic acid: implications for mechanisms of apoptosis.

Internucleosomal fragmentation of DNA, the most widely used biochemical indicator of apoptosis, is believed to contribute to loss of viability because the nuclease inhibitor, aurintricarboxylic acid, delays or prevents cell death in a range of experimental systems. We report here that auritricarboxylic acid inhibits topoisomerase II in vitro, the concentration required (< or = 0.2 microM) being less than that usually employed in studies of apoptosis. Since topoisomerase II mediates chromatin condensation during apoptosis, the efficacy of ATA in preventing or delaying cell death may not be the result of nuclease inhibition.