Novel Transgenic Mouse Model for Studying Human Serum Albumin as a Biomarker of Carcinogenic Exposure.

Albumin is a commonly used serum protein for studying human exposure to xenobiotic compounds, including therapeutics and environmental pollutants. Often, the reactivity of albumin with xenobiotic compounds is studied ex vivo with human albumin or plasma/serum samples. Some studies have characterized the reactivity of albumin with chemicals in rodent models; however, differences between the orthologous peptide sequences of human and rodent albumins can result in the formation of different types of chemical-protein adducts with different interaction sites or peptide sequences. Our goal is to generate a human albumin transgenic mouse model that can be used to establish human protein biomarkers of exposure to hazardous xenobiotics for human risk assessment via animal studies. We have developed a human albumin transgenic mouse model and characterized the genotype and phenotype of the transgenic mice. The presence of the human albumin gene in the genome of the model mouse was confirmed by genomic PCR analysis, whereas liver-specific expression of the transgenic human albumin mRNA was validated by RT-PCR analysis. Further immunoblot and mass spectrometry analyses indicated that the transgenic human albumin protein is a full-length, mature protein, which is less abundant than the endogenous mouse albumin that coexists in the serum of the transgenic mouse. The transgenic protein was able to form ex vivo adducts with a genotoxic metabolite of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine, a procarcinogenic heterocyclic aromatic amine formed in cooked meat. This novel human albumin transgenic mouse model will facilitate the development and validation of albumin-carcinogen adducts as biomarkers of xenobiotic exposure and/or toxicity in humans.

Subcellular location and molecular mobility of human cytosolic sulfotransferase 1C1 in living human embryonic kidney 293 cells.

Cytosolic sulfotransferases were first isolated from the hepatic cytosol, and they have been localized in the cytoplasm of formaldehyde-fixed human cell samples. The current work was carried out to determine the subcellular localization and molecular mobility of cytosolic sulfotransferases in living human embryonic kidney (HEK) 293 cells. In this work, the subcellular location of human cytosolic sulfotransferase 1C1 (SULT1C1) was studied in cultured HEK293 cells using confocal laser-scanning microscopy. A green fluorescent protein (GFP)-tagged SULT1C1 protein was localized in the cytoplasm of living HEK293 cells. This is consistent with results from previous studies on several other cytosolic sulfotransferase isoforms. Fluorescence recovery after photobleaching microscopy was performed to assess the molecular mobility of the expressed GFP-SULT1C1 molecules. The results suggested that the expressed recombinant GFP-SULT1C1 molecules in living HEK293 cells may include both mobile and immobile populations. To obtain additional insights into the subcellular location of SULT1C1, two machine learning algorithms, Sequential Minimal Optimization and Multilayer Perceptron, were used to compute the probability distribution for the localization of SULT1C1 in nine selected cellular compartments. The resulting probability distribution suggested that the most likely subcellular location of SULT1C1 is the cytosol.

Non ECG gated supine to prone left ventricular volume ratio: a novel marker for myocardial ischemia.

Transient ischemic dilation (TID), a marker of severe coronary artery disease (CAD), is the post-stress to rest left ventricular (LV) volume ratio quantified using non ECG gated single photon emission computerized tomography (SPECT). Although prone positioning causes physiological reduction of LV volume in normal subjects, we hypothesize this may not occur in TID with underlying severe CAD as cardiac hemodynamics worsen when prone. We aim to evaluate the utility of the non ECG gated supine to prone LV volume ratio (SPLVr) for identifying severe CAD. Retrospective data analysis from 130 patients with TID ratio ≥ 1.21 and both post-stress supine and prone images. SPLVr had a significant negative correlation with summed stress (r = - 0.221, p = 0.011) and rest (r = - 0.292, p = 0.001) scores. Of the 129 cases with follow-up invasive or computed tomography coronary angiography, 52 (40.3%) had severe CAD (left main ≥ 50% stenosis, 3-vessel with ≥ 70% stenosis or 2-vessel with proximal left anterior descending ≥ 70% stenosis). Mean SPLVr was significantly lower in severe CAD cases (1.05 ± 0.14 vs 1.12 ± 0.17, p = 0.012). SPLVr predicted severe CAD on univariate [OR 0.12 (95% CI 0.00-0.35) p = 0.01] but not in multivariate analysis. SPLVr is a novel marker that negatively correlates with extent of perfusion abnormalities and is lower amongst TID patients with severe CAD. Larger studies are needed to assess if SPLVr can reliably identify underlying severe CAD amongst TID cases.

Transfection of human prostate cancer CA-HPV-10 cells with cytosolic sulfotransferase SULT1E1 affects estrogen signaling and gene transcription.

Human cytosolic sulfotransferase SULT1E1 catalyzes the sulfation of estrogens and estrogenic drugs in human reproductive tissues. Logically, this estrogen-preferring sulfotransferase isoform could play a regulatory role in estrogen signaling activities in human reproductive cells, including the prostate cells. This hypothesis was tested using DNA microarray and real-time reverse transcription-polymerase chain reaction methods in the present work. Potential changes in the transcriptional expression of selected signal transduction-related genes in human prostate cancer CA-HPV-10 cell line after SULT1E1 transfection were examined by DNA microarray methods. Notable changes were observed in the mRNA expression levels of TFRC, a cell membrane transferrin receptor gene, and TMEPAI, a gene encoding a steroid-dependent mRNA product. Expression of TFRC was down-regulated, whereas expression of TMEPAI was up-regulated by SULT1E1 transfection in CA-HPV-10 cells. Data from the current studies also showed that the estrogen-induced estrogen response element activation in CA-HPV-10 cells was repressed after the cells were transfected with SULT1E1. These results indicate that SULT1E1 may function as a transcriptional mediator in human prostate cancer CA-HPV-10 cells.

Cellular localization studies on human estrogen sulfotransferase SULT1E1 in human embryonic kidney 293 cells.

Human cytosolic sulfotransferase SULT1E1 catalyzes the sulfation of endogenous estrogens as well as xenobiotic estrogen-like chemicals. This reaction increases the water solubility of the molecule, which may affect its cellular distribution and biological activity. This could alter estrogen signaling to the estrogen receptor in human estrogen receptor-positive cells. The current work characterized the cellular distribution of SULT1E1 in the human embryonic kidney 293 (HEK293) cell line using green fluorescent protein (GFP) tagging and immunochemistry methods. The GFP-tagged recombinant SULT1E1 protein was expressed and localized in the cytoplasm of HEK293 cells. By using a commercial anti-SULT1E1 peptide antibody, a 35.7-kDa protein was detected in HEK293 cells via Western blot. The molecular mass of the protein detected suggested that it may be related to native SULT1E1 protein. However, reverse transcription-polymerase chain reaction (RT-PCR) with gene-specific primers could not confirm the presence of the SULT1E1 transcript in the total RNA sample of HEK293 cells. The discrepancy between protein and transcript data could be due to the instability of SULT1E1 mRNA or the specificity of the anti-SULT1E1 antibody used. In the present work, RT-PCR analysis with gene-specific primers also identified a transcript fragment of human estrogen-related receptor gamma. Future studies on the functional relationship between estrogen-related receptors and sulfotransferases are expected to provide additional insights into the physiological and toxicological roles of human estrogen sulfotransferases.

Enantioselectivity of human hydroxysteroid sulfotransferase ST2A3 with naphthyl-1-ethanols.

Hydroxysteroid (alcohol) sulfotransferases catalyze the sulfation of several endogenous steroids and many hydrophobic xenobiotic alcohols. The substrate stereoselectivities of sulfotransferases may be critically important in determining their overall roles in metabolism of drugs, carcinogens, and other xenobiotics. In the present work, stereoselectivity of the human hydroxysteroid sulfotransferase ST2A3 (also variously named as SULT2A1 or human DHEA-ST) was examined through analysis of its catalytic activities with the enantiomers of 1-naphthyl-1-ethanol and 2-naphthyl-1-ethanol. The kcat/Km value for sulfation of the R-(+)-enantiomer of 1-naphthyl-1-ethanol catalyzed by ST2A3 was 3.3 min-1mM-1, whereas the S-(-)-enantiomer was not a substrate for the enzyme. S-(-)-1-naphthyl-1-ethanol did however interact with ST2A3 as an inhibitor of the sulfation of dehydroepiandrosterone. This substrate stereospecificity was not present with the enantiomers of 2-naphthyl-1-ethanol, since both were substrates for the enzyme. Such differences between the sulfation of 1- and 2-naphthyl-1-ethanol are consistent with the importance of steric interactions between the ethanol group and a hydrogen atom at the peri-position (C8) on the naphthyl ring in 1-naphthyl-1-ethanol that combine with the topology of the enzyme's active site to determine stereospecificity.

Influence of phenylalanines 77 and 138 on the stereospecificity of aryl sulfotransferase IV.

Aryl sulfotransferase (AST) IV (also named tyrosine-ester sulfotransferase and ST1A1) is a major phenol sulfotransferase in the rat, and it catalyzes the sulfation of many drugs, carcinogens, and other xenobiotics that contain phenol, benzylic alcohol, N-hydroxy arylamine, and oxime functional groups. Previous work discovered a stereospecificity of AST IV toward the enantiomers of 1,2,3,4-tetrahydro-1-naphthol and varying degrees of stereoselectivity with other chiral benzylic alcohols. The studies described here were directed toward understanding the roles of specific amino acid residues at the substrate binding site in determining the stereoselectivity of this sulfotransferase isoform. Docking experiments with a homology model of AST IV revealed three amino acid residues, Phe77, Phe138, and Tyr236, that may potentially be important for interactions with substituents on the chiral carbon of a benzylic alcohol serving as a sulfuryl acceptor, thereby imparting stereoselectivity. To test this hypothesis, mutants were constructed wherein each of the above residues was substituted with alanine. Kinetic studies on the sulfation of the enantiomers of 1,2,3,4-tetrahydro-1-naphthol indicated that the stereospecificity of the sulfotransferase was altered by the substitutions of alanine for either Phe77 or Phe138, but stereospecificity was maintained by alanine substitution at Tyr236. Molecular models of the mutant enzymes interacting with enantiomers of 1,2,3,4-tetrahydro-1-naphthols and with 2-naphthol indicate that Phe77 and Phe138 provide significant steric interactions at the active site that both enhance catalytic efficiency and impart stereospecificity in molecular recognition of substrates and inhibitors.