Exposure to 4-aminobiphenyl (4-ABP), an environmental and tobacco smoke carcinogen that targets the bladder urothelium, leads to DNA adduct formation and cancer development [1]. Two major analytical challenges in DNA adduct analysis of human samples have been limited sample availability and the need to reach detection limits approaching the part-per-billion threshold. By operating at nano-flow rates and incorporating a capillary analytical column in addition to an online sample enrichment step, we have developed a sensitive and quantitative HPLC–MS/MS method appropriate for the analysis of such samples. This assay for the deoxyguanosine adduct of 4-ABP (dG-C8-4-ABP) gave mass detection limits of 20 amol in 1.25 μg of DNA (5 adducts in 109 nucleosides) with a linear range of 70 amol to 70 fmol. 4-ABP-exposed human bladder cells and rat bladder tissue were analyzed in triplicate, and higher dose concentrations led to increased numbers of detected adducts. It was subsequently established that sample requirements could be further reduced to 1 μg digestions and the equivalent of 250 ng DNA per injection for the detection of low levels of dG-C8-4-ABP in a matrix of exfoliated human urothelial cell DNA. This method is appropriate for the characterization and quantification of DNA adducts in human samples and can lead to a greater understanding of their role in carcinogenesis and also facilitate evaluation of chemopreventive agents.
Neuronopathic glycosphingolipidoses are a sub-group of lysosomal storage disorders for which there are presently no effective therapies. Here, we evaluated the potential of substrate reduction therapy (SRT) using an inhibitor of glucosylceramide synthase (GCS) to decrease the synthesis of glucosylceramide (GL1) and related glycosphingolipids. The substrates that accumulate in Sandhoff disease (e.g., ganglioside GM2 and its nonacylated derivative, lyso-GM2) are distal to the drug target, GCS. Treatment of Sandhoff mice with a GCS inhibitor that has demonstrated CNS access (Genz-682452) reduced the accumulation of GL1 and GM2, as well as a variety of diseaseassociated substrates in the liver and brain. Concomitant with these effects was a significant decrease in the expression of CD68 and glycoprotein non-metastatic melanoma B protein (Gpnmb) in the brain, indicating a reduction in microgliosis in the treated mice. Moreover, using in vivo imaging, we showed that the monocytic biomarker translocator protein (TSPO), which was elevated in Sandhoff mice, was normalized following Genz-682452 treatment. These positive effects translated in turn into a delay ($28 days) in loss of motor function and coordination, as measured by rotarod latency, and a significant increase in longevity ($17.5%). Together, these results support the development of SRT for the treatment of gangliosidoses, particularly in patients with residual enzyme activity.
SLC6A19 (B0AT1) is a neutral amino acid transporter, the loss of function of which results in Hartnup disease. SLC6A19 is also believed to have an important role in amino acid homeostasis, diabetes, and weight control. A small-molecule inhibitor of human SLC6A19 (hSLC6A19) was identified using two functional cell-based assays: a fluorescence imaging plate reader (FLIPR) membrane potential (FMP) assay and a stable isotope-labeled neutral amino acid uptake assay. A diverse collection of 3440 pharmacologically active compounds from the Microsource Spectrum and Tocriscreen collections were tested at 10 µM in both assays using MDCK cells stably expressing hSLC6A19 and its obligatory subunit, TMEM27. Compounds that inhibited SLC6A19 activity in both assays were further confirmed for activity and selectivity and characterized for potency in functional assays against hSLC6A19 and related transporters. A single compound, cinromide, was found to robustly, selectively, and reproducibly inhibit SLC6A19 in all functional assays. Structurally related analogs of cinromide were tested to demonstrate structure–activity relationship (SAR). The assays described here are suitable for carrying out high-throughput screening campaigns to identify modulators of SLC6A19.
Nrf2 is a major cytoprotective gene and is a key chemopreventive target against cancer and other diseases. Here we show that Nrf2 faces a dilemma in defense against 4-aminobiphenyl (ABP), a major human bladder carcinogen from tobacco smoke and other environmental sources. While Nrf2 protected mouse liver against ABP (which is metabolically activated in liver), the bladder level of N-(deoxyguanosin-8-yl)-4-aminobiphenyl (dG-C8-ABP), the predominant ABP-DNA adduct formed in bladder cells and tissues, was markedly higher in Nrf2+/+ mice than in Nrf2−/− mice after ABP exposure. Notably, Nrf2 protected bladder cells against ABP in vitro. Mechanistic investigations showed that the dichotomous effects of Nrf2 could be explained at least partly by upregulation of UDP-glucuronosyltransferase (UGT). Nrf2 promoted conjugation of ABP with glucuronic acid in the liver, increasing urinary excretion of the conjugate. While glucuronidation of ABP and its metabolites is a detoxification process, these conjugates, which are excreted in urine, are known to be unstable in acidic urine, leading to delivery of the parent compounds to bladder. Hence, while higher liver UGT activity may protect the liver against ABP it increases bladder exposure to ABP. These findings raise concerns of potential bladder toxicity when Nrf2-activating chemopreventive agents are used in humans exposed to ABP, especially in smokers. We further demonstrate that 5,6-dihydrocyclopenta[c][1,2]-dithiole-3(4H)-thione (CPDT) significantly inhibits dG-C8-ABP formation in bladder cells and tissues, but does not appear to significantly modulate ABP-catalyzing UGT in liver. Thus, CPDT exemplifies a counteracting solution to the dilemma posed by Nrf2.
Crystalline europium bromate, [Eu(H 2 O) 9 ][(BrO 3 ) 3 ], europium ethyl sulfate, [Eu(H 2 O) 9 ][(C 2 H 5 SO 4 ) 3 ], and europium decavanate, [Eu(H 2 O) 8 ] 2 [(V 10 O 28 )]‚8H 2 O, are used as models for nine-and eight-coordinate Eu 3+ aquo species in solution. A comparison of the 5 D 1 r 7 F 0 laser excitation spectra and D 0 f 7 F 1,2 emission spectra of the model crystal systems with that of 0.1 M EuCl 3 (aq) clearly indicates a significant presence of the Eu(H 2 O) 8 3+ species in aqueous solution. Modeling the 0.1 M EuCl 3 (aq) spectra using weighted sums of the eight and nine-coordinate crystal spectra suggest that approximately two-thirds of Eu 3+ ions exist as Eu(H 2 O) 8 3+ in aqueous solution. The current study takes advantage of the symmetry of the Eu(H 2 O) n 3+ complexes and lends further support, from this perspective, that an equilibrium exists between Eu(H 2 O) 8 3+ and Eu(H 2 O) 9 3+ , with significant representation from both species.
NF-E2 related factor 2 (Nrf2) plays a pivotal role in cytoprotection against carcinogens and oxidants. Activating Nrf2 has been widely suggested as a key chemopreventive strategy against cancer and other diseases. Here, we show that Nrf2 faces a serious dilemma in dealing with 4-aminobiphenyl (ABP), a major human bladder carcinogen from tobacco smoke and other sources. ABP is metabolized/activated primarily in the liver. While Nrf2 significantly protected mouse liver against ABP in vivo, bladder DNA damage was approximately 2.6 fold higher in Nrf2+/+ mice than in Nrf2-/- mice after ABP exposure, as measured by dG-C8-ABP, the predominant ABP-DNA adduct formed in bladder cells and tissues. Interestingly, Nrf2 significantly protected bladder cells against ABP in vitro. Subsequent experiments revealed that UDP-glucuronosyltransferase (UGT) might be the principal, if not the only mediator of the dichotomous effects of Nrf2. Nrf2 upregulated UGT, promoted glucuronide conjugation of ABP in the liver, and increased urinary excretion of the conjugate. While glucuronidation of ABP and its metabolites is generally a detoxification process, these conjugates, which are excreted in urine, are known to be unstable in acidic urine and deliver the parent compounds to bladder. Hence, higher liver UGT activity may protect liver against ABP but increases bladder exposure to ABP. We further show that 5,6-dihydrocyclopenta[c][1,2]-dithiole-3(4H)-thione (CPDT), a promising cancer chemopreventive dithiolethione, was able to significantly activate Nrf2 cytoprotective signaling and to inhibit dG-C8-ABP formation in the bladder in vivo, but did not modulate liver UGT expression. Thus, CPDT exemplifies a counteracting solution to the dilemma faced by Nrf2 with regard to ABP. In conclusion, our study shows that Nrf2 increases ABP genotoxicity in the bladder in vivo and that UGT may be the key mediator. Using CPDT, we also demonstrate how the adverse effect of Nrf2 may be mitigated. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1843. doi:10.1158/1538-7445.AM2011-1843
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