Adenosinergic regulation of striatal clock gene expression and ethanol intake during constant light.

Circadian rhythm and sleep disruptions occur frequently in individuals with alcohol use disorders (AUD) and present significant barriers to treatment. Recently, a variant of adenosine transporter, equilibrative nucleoside transporter 1 (ENT1), was associated with the co-occurrence of sleep problems and AUD. We have previously shown that mice lacking ENT1 (ENT1 KO) have reduced adenosine levels in the striatum and drink more alcohol compared with wild types (WT). However, it is unknown whether ENT1 deletion disrupts circadian rhythms, which may contribute to alcohol preference in ENT1 KO mice. Here we used these mice to determine whether endogenous adenosine regulates circadian genetic and behavioral rhythms and influences alcohol intake during chronodisruption. We examined circadian locomotor activity in ENT1 KO vs WT littermates and found that ENT1 KO mice were both active earlier and hyperactive compared with WT mice at night. We used real-time PCR and immunohistochemistry to estimate striatal clock gene levels and found that PER2 expression in the striatum was blunted by ENT1 deletion or A2A receptor (A2AR) antagonism. Next, we exposed ENT1 KO and WT mice to constant light (LL) and found further elevation in ethanol intake in ENT1 KO, but not in WT mice, supporting the notion that circadian dysfunction may contribute to increased alcohol intake in ENT1 KO mice. Finally, we showed that A2AR agonist administration normalized PER1 and PER2 expression and circadian locomotor activity in ENT1 KO mice. Together, our results demonstrate that adenosine signaling regulates cellular and behavioral circadian timing and influences alcohol intake during chronodisruption.

Correlation of CYP2B6, CYP2C19, ABCC4 and SOD2 genotype with outcomes in allogeneic blood and marrow transplant patients.

CYP2B6, CYP2C19, ABCC4, and SOD2 have been implicated in adverse drug reactions and survival after cyclophosphamide (CPA) treatment. 110 BMT patients who received high dose CPA treatment were genotyped for variants in these genes and the results were correlated with toxicity and relapse. CYP2B6 genotype significantly influenced overall toxicity suggesting active CYP2B6 alleles led to higher rates of overall toxicity. The p.R487C deficiency allele was significantly associated with a lower rate of overall toxicity and a higher rate of relapse. SOD2 rs4880 V16A polymorphism was associated with significantly less CPA-related overall toxicity and significantly lower relapse rates by Kaplan-Meier analysis although the SOD2 finding regarding relapse was not significant when evaluated by the cumulative incidence function.

Sex-Specific Regulation of Depression, Anxiety-Like Behaviors and Alcohol Drinking in Mice Lacking ENT1.

OBJECTIVES: Adenosine signaling has been implicated in the pathophysiology of several psychiatric disorders including alcoholism, depression, and anxiety. Adenosine levels are controlled in part by transport across the cell membrane by equilibrative nucleoside transporters (ENTs). Recent evidence showed that a polymorphism in the gene encoding ENT1 is associated with comorbid depression and alcoholism in women. We have previously shown that deletion of ENT1 reduces ethanol intoxication and elevates alcohol intake in mice. Interestingly, ENT1 null mice display decreased anxiety-like behavior compared to wild-type littermates. However, our behavioral studies were performed only in male mice. Here, we extend our research to include female mice, and test the effect of ENT1 knockout on other behavioral correlates of alcohol drinking, including depressive and compulsive behavior, in mice. METHODS: To assess depression-like behavior, we used a forced swim test modified for mice. We examined anxiety-like behavior and locomotor activity in open field chambers, and perseverant behavior using the marble-burying test. Finally, we investigated alcohol consumption and preference in female mice using a two-bottle choice paradigm. RESULTS: ENT1 null mice of both sexes showed reduced immobility time in the forced swim test and increased time in the center of the open field compared to wild-type littermates. ENT1 null mice of both sexes showed similar locomotor activity levels and habituation to the open field chambers. Female ENT1 null mice displayed increased marble-burying compared to female wild-types, but no genotype difference was evident in males. Female ENT1 null mice showed increased ethanol consumption and preference compared to female wild-types. CONCLUSIONS: Our findings suggest that ENT1 contributes to several important behaviors involved in psychiatric disorders. Inhibition of ENT1 may be beneficial in treating depression and anxiety, while enhancement of ENT1 function may reduce compulsive behavior and drinking, particularly in females.

Characterization of large rearrangements in autosomal dominant polycystic kidney disease and the PKD1/TSC2 contiguous gene syndrome.

Large DNA rearrangements account for about 8% of disease mutations and are more common in duplicated genomic regions, where they are difficult to detect. Autosomal dominant polycystic kidney disease (ADPKD) is caused by mutations in either PKD1 or PKD2. PKD1 is located in an intrachromosomally duplicated region. A tuberous sclerosis gene, TSC2, lies immediately adjacent to PKD1 and large deletions can result in the PKD1/TSC2 contiguous gene deletion syndrome. To rapidly identify large rearrangements, a multiplex ligation-dependent probe amplification assay was developed employing base-pair differences between PKD1 and the six pseudogenes to generate PKD1-specific probes. All changes in a set of 25 previously defined deletions in PKD1, PKD2 and PKD1/TSC2 were detected by this assay and we also found 14 new mutations at these loci. About 4% of the ADPKD patients in the CRISP study were found to have gross rearrangements, and these accounted for about a third of base-pair mutation negative families. Sensitivity of the assay showed that about 40% of PKD1/TSC contiguous gene deletion syndrome families contained mosaic cases. Characterization of a family found to be mosaic for a PKD1 deletion is discussed here to illustrate family risk and donor selection considerations. Our assay improves detection levels and the reliability of molecular testing of patients with ADPKD.

The metabolism of pyrazoloacridine (NSC 366140) by cytochromes p450 and flavin monooxygenase in human liver microsomes.

Pyrazoloacridine (PZA) is an experimental antitumor agent presently under investigation for treatment of solid tumors on the basis of its unique mechanism of action and selectivity for human solid tumor xenograft in mice. Using capillary electrophoresis coupled with electrospray ionization mass spectrometry, we have identified three oxidative PZA metabolites, 9-desmethyl-PZA, N-demethyl-PZA, and PZA N-oxide. The cytochrome p450 (CYP) isoforms involved in PZA metabolism were characterized by studies with CYP chemical inhibitors, correlation of marker activities for selected CYPs with formation of the metabolites using a human liver panel, and PZA metabolism by cDNA-expressed CYPs. 9-Desmethyl-PZA formation was catalyzed by CYP1A2, whereas N-demethyl-PZA formation was catalyzed by CYP3A4. PZA N-oxide formation was catalyzed by flavin monooxygenase (FMO) rather than CYP, as determined by studies with chemical inhibitors of FMO and metabolism by cDNA-expressed human flavin monooxygenase. After administration of [10b-(14)C]PZA to mice, six urinary metabolites were detected by high-performance liquid chromatography UV and radiochromatograms including 9-desmethyl-PZA, N-demethyl-PZA, and PZA N-oxide. Trace concentrations of 9-desmethyl-PZA and PZA N-oxide were detected in mouse plasma. PZA N-oxide and N-demethyl-PZA were detected in urine from patients after PZA administration. PZA, 9-desmethyl-PZA, and PZA N-oxide inhibited growth of A375 human melanoma cells. IC(50) values were 0.17, 0.11, and 7.0 micro M, respectively, for the three molecules.