Transport of A1 adenosine receptor agonist tecadenoson by human and mouse nucleoside transporters: evidence for blood-brain barrier transport by murine equilibrative nucleoside transporter 1 mENT1.

The high density of A1 adenosine receptors in the brain results in significant potential for central nervous system (CNS)-related adverse effects with A1 agonists. Tecadenoson is a selective A1 adenosine receptor agonist with close similarity to adenosine. We studied the binding and transmembrane transport of tecadenoson by recombinant human equilibrative nucleoside transporters (hENTs) hENT1 and hENT2, and human concentrative nucleoside transporters (hCNTs) hCNT1, hCNT2, and hCNT3 in vitro and by mouse mENT1 in vivo. Binding affinities of the five recombinant human nucleoside transporters for tecadenoson differed (hENT1 > hCNT1 > hCNT3 > hENT2 > hCNT2), and tecadenoson was transported largely by hENT1. Pretreatment of mice with a phosphorylated prodrug of nitrobenzylmercaptopurine riboside, an inhibitor of mENT1, significantly decreased brain exposure to tecadenoson compared with that of the untreated (control) group, suggesting involvement of mENT1 in transport of tecadenoson across the blood-brain barrier (BBB). In summary, ENT1 was shown to mediate the transport of tecadenoson in vitro with recombinant and native human protein and in vivo with mice. The micromolar apparent Km value of tecadenoson for transport by native hENT1 in cultured cells suggests that hENT1 will not be saturated at clinically relevant (i.e., nanomolar) concentrations of tecadenoson, and that hENT1-mediated passage across the BBB may contribute to the adverse CNS effects observed in clinical trials. In contrast, in cases in which a CNS effect is desired, the present results illustrate that synthetic A1 agonists that are transported by hENT1 could be used to target CNS disorders because of enhanced delivery to the brain.

Differential cytotoxic effects of arsenic compounds in human acute promyelocytic leukemia cells.

Arsenic trioxide, As(2)O(3), has successfully been used to treat acute promyelocytic leukemia (APL). Induction of apoptosis in cancerous cells has been proposed to be the underlying mechanism for the therapeutic efficacy of arsenic. To further understand the cytotoxicity of arsenic compounds in APL cells, HL-60 cells were exposed to graded concentrations of the following arsenicals for up to 48 h: arsenic trioxide (As(III)), sodium arsenate (As(V)), phenylarsine oxide (PAO(III)), monomethylarsonous acid (MMA(III)), monomethylarsonic acid (MMA(V)) and dimethylarsinic acid (DMA(V)), and the viability and modes of cell death assessed. The arsenic-exposed cells were stained with annexin V-PE and 7-aminoactinomycin D (7-AAD) and analyzed by flow cytometry in order to detect apoptotic and viable cells while cell morphology was visualized using scanning and transmission electron microscopy. Acridine orange staining and microtubule-associated protein 1 light chain 3 (MAP-LC3) detection were used to recognize autophagic cell death. The results showed that the compounds reduced viable HL-60 cells by inducing apoptosis in a concentration-dependent manner. None of the compounds tested caused a significant change in binding of acridine orange or redistribution of MAP-LC3. Potencies of the six different arsenic compounds tested were ranked as PAO(III)>MMA(III)> or =As(III)>As(V)>MMA(V)>DMA(V). An increase in caspase-3 activity by PAO(III), MMA(III) and DMA(V) implied that these compounds induced apoptosis in HL-60 cells through a caspase-dependent mechanism, but the other arsenic compounds failed to activate caspase-3, suggesting that they induce apoptosis by an alternative pathway.

Reliability of using urinary and blood trichloroacetic acid as a biomarker of exposure to chlorinated drinking water disinfection byproducts.

This study was designed to analyse the reliability of using urinary and blood trichloroacetic acid (TCAA) as a biomarker of exposure. A total of 46 healthy women consumed supplied TCAA-containing tap water for 15 days and provided urine and blood samples for TCAA measurements. The findings revealed that the reliability of measurements was excellent by using measures of TCAA ingestion, blood concentration and urinary excretion (intraclass correlation coefficients (ICC) > 0.75, p < 0.001). Volume of tap water consumption (ICC = 0.69) and creatinine-adjusted urinary concentration (ICC = 0.72) were less reliable. This indicated that the intraindividual variability was small and the interindividual reliability was high by using these measures in this cohort study. Laboratory variability did not significantly contribute to total variance (ICC > 0.95, p < 0.001). Other possible sources of variation such as bathing, showering, dishwashing and physical activities were unlikely to contribute significantly to total variance. For sampling strategies, 1-day blood sampling and 2-day urine sampling are sufficient to achieve reliability for an epidemiological study if a quasi-steady-state TCAA level in the body is reached. The results suggest that TCAA ingestion, TCAA loading in blood and urinary TCAA excretion are reliable measures for use as biomarkers in epidemiological studies.

Validation of urinary trichloroacetic acid as a biomarker of exposure to drinking water disinfection by-products.

Disinfection by-products (DBPs) in drinking water represent a public health issue and a challenge for epidemiology to provide evidence towards the causation of various hypothesized health effects. Validation of a biomarker of exposure to DBPs is a strategy to achieve progress which has been advocated. The objective of this study was to validate urinary trichloroacetic acid (TCAA) excretion as a biomarker of exposure to DBPs in an experimental exposure cohort. A total of 52 healthy women participated in the study. Participants consumed supplied tap water for 15 d and provided urine and blood samples for TCAA measurements. The findings revealed that (1) background levels of TCAA in urine and blood were readily detectable, (2) TCAA levels in blood and urine increased with increased amounts of TCAA ingested, (3) the correlations between measurements of TCAA ingestion and urinary excretion were modest (r=0.66, p<0.001) based on one days' sampling and high (r=0.77-0.83, p<0.001) based on two to four days' sampling, (4) the correlations between measurements of TCAA ingestion and blood TCAA concentration were high (r=0.80, p<0.001) and (5) multiple days' urinary TCAA measures improved the prediction of TCAA ingestion through urinary TCAA excretion. TCAA can be a valid biomarker of exposure for DBPs in drinking water.

Biodistribution and uptake of 3'-deoxy-3'-fluorothymidine in ENT1-knockout mice and in an ENT1-knockdown tumor model.

UNLABELLED: (18)F-3'-Deoxy-3'-fluorothymidine ((18)F-FLT) is a PET tracer that accumulates in proliferating tissues. The current study was undertaken to determine whether equilibrative nucleoside transporter 1 (ENT1) is important for (18)F-FLT uptake in normal tissues and tumors. METHODS: ENT1-knockout (ENT1(-/-)) mice were generated and compared with wild-type (ENT1(+/+)) mice using small-animal (18)F-FLT PET. In addition, ENT1(+/+) mice were also injected with the ENT1 inhibitor nitrobenzylmercaptopurine ribonucleoside phosphate (NBMPR-P) at 1 h before radiotracer injection, followed by (18)F-FLT small-animal PET. Tissues of interest were analyzed for thymidine kinase 1 and nucleoside transporters by immunoblotting and immunohistochemistry, respectively, and plasma thymidine levels were analyzed by liquid chromatography-mass spectrometry. Human lung carcinoma A549 cells were stably transfected with pSUPER-producing short-hairpin RNA against human ENT1 (hENT1) or a scrambled sequence with no homology to mammalian genes (A549-pSUPER-hENT1 and A549-pSUPER-SC, respectively). Cultured transfected cells were characterized for hENT1 transcript levels and (18)F-FLT uptake using real-time polymerase chain reaction and (3)H-FLT uptake assays, respectively. Transfected A549 cells were grown as xenograft tumors in NIH-III mice, which were analyzed by (18)F-FLT small-animal PET. RESULTS: Compared with noninjected ENT1(+/+) mice, ENT1(+/+) mice injected with NBMPR-P and ENT1(-/-) mice displayed a reduced percentage injected dose per gram (%ID/g) for (18)F-FLT in the blood (84 and 81%, respectively) and an increased %ID/g for (18)F-FLT in the spleen (188 and 469%, respectively) and bone marrow (266 and 453%, respectively). ENT1(-/-) mice displayed 1.65-fold greater plasma thymidine levels than did ENT1(+/+) mice. Spleen tissue from ENT1(+/+) and ENT1(-/-) mice displayed similar thymidine kinase 1 protein levels and significant concentrative nucleoside transporter 1 and 3 staining. Compared with A549-pSUPER-SC cells, A549-pSUPER-hENT1 cells displayed 0.45-fold hENT1 transcript levels and 0.68-fold (3)H-FLT uptake. Compared with A549-pSUPER-SC xenograft tumors, A549-pSUPER-hENT1 xenograft tumors displayed 0.76-fold %ID/g values (ex vivo gamma-counts) and 0.65-fold maximum standardized uptake values (PET image analysis) for (18)F-FLT uptake at 1 h after tracer injection. CONCLUSION: Loss of ENT1 activity significantly affected (18)F-FLT biodistribution in mice and (18)F-FLT uptake in xenograft tumors, suggesting that nucleoside transporters are important mediators of (18)F-FLT uptake in normal and transformed cells.

Cytotoxicity of 2-chlorodeoxyadenosine and arabinosylcytosine in leukaemic lymphoblasts from paediatric patients: significance of cellular nucleoside transporter content.

2-chlorodeoxyadenosine (2-CdA) and arabinosylcytosine (araC) are nucleoside drugs that are used to treat various leukaemias, although 2-CdA has not been tested extensively in children with acute lymphoblastic leukaemia (ALL). Nucleoside cytotoxicity depends on the conversion of these agents to 5'-phosphate derivatives, following drug entry into cells via nucleoside transport (NT) processes. This study compared es nucleoside transporter content, determined using a flow cytometric assay with SAENTA [5'-S-(2-aminoethyl)-N6-(4-nitrobenzyl)-5'-thioadenosine] fluorescein, and cytotoxicities of 2-CdA and araC in fresh lymphoblasts from previously untreated paediatric ALL patients and the human T-lymphoblast cell line, CCRF-CEM. Lymphoblast samples from individual patients ranged widely in sensitivity to both 2-CdA (IC50, 6 nmol/l to > 5 micromol/l; mean = 418 nmol/l; n = 8) and araC (IC50, 59 nmol/l to > 5 micromol/l; mean = 1050 nmol/l; n = 7), although IC50 values for the two drugs were correlated (r = 0.78, P = 0.032, n = 7). Cellular es nucleoside transporter content varied more than 35-fold among samples from 10 patients. The correlation between es nucleoside transporter content and drug sensitivity was statistically significant for araC (r = -0.93, P = 0.023, n = 5), but not for 2-CdA (r = -0.57, P = 0.23, n = 6). Exposure of CCRF-CEM cells to araC resulted in a substantial araC concentration-dependent increase in the relative survival of es transporter-deficient cells, whereas the increase was slight following exposure to 2-CdA. We conclude that, in ALL lymphoblasts, es nucleoside transporter content is a determinant of araC sensitivity and that a deficiency in NT may impart resistance to araC.