Enzymatic sulfation of tocopherols and tocopherol metabolites by human cytosolic sulfotransferases

Tocopherols are essential micronutrients for mammals widely known as potent lipid-soluble antioxidants that are present in cell membranes. Recent studies have demonstrated that most of the carboxychromanol (CEHC), a tocopherol metabolite, in the plasma exists primarily in sulfate- and glucuronide-conjugated forms. To gain insight into the enzymatic sulfation of tocopherols and their metabolites, a systematic investigation was performed using all 14 known human cytosolic sulfotransferases (SULTs). The results showed that the members of the SULT1 family displayed stronger sulfating activities toward tocopherols and their metabolites. These enzymes showed a substrate preference for γ-tocopherol over α-tocopherol and for γ-CEHC over other CEHCs. Using A549 human lung epithelial cells in a metabolic labeling study, a similar trend in the sulfation of tocopherols and CEHCs was observed. Collectively, the results obtained indicate that SULT-mediated enzymatic sulfation of tocopherols and their metabolites is a significant pathway for regulation of the homeostasis and physiological functions of these important compounds.     

Sulfation of tibolone metabolites by human postmenopausal liver and small intestinal sulfotransferases (SULTs)

Sulfation is a major pathway in humans for the biotransformation of steroid hormones and structurally related therapeutic agents. Tibolone is a synthetic steroid used for the treatment for climacteric symptoms and postmenopausal osteoporosis. Sulfation inactivates the hydroxylated metabolites, 3alpha-hydroxytibolone (3alpha-OH-tibolone) and 3beta-hydroxytibolone (3beta-OH-tibolone), and contributes to the regulation of tissue responses to tibolone. We detected SULT1A1, SULT1A3, SULT1E1 and SULT2A1 mRNA expression by RT-PCR in postmenopausal liver and small intestine. Liver pool (n=5) SULT activities measured with tibolone substrates reflected COS-1 expressed SULT2A1 and SULT1E1 activities. Liver SULT2A1 activity (1.8 +/- 0.3 units/mg protein, n = 8, mean +/- SEM), and activities with 3alpha-OH-tibolone (0.6 +/- 0.1, n = 8) and 3beta-OH-tibolone (0.9 +/- 0.2, n = 8) were higher than SULT1E1 activities (<0.05, n = 10). SULT1E1 activities were low or not detected in many samples. Mean small intestinal activities were 0.03 +/- 0.01 with 3alpha-OH-tibolone and 0.04 +/- 0.01 with 3beta-OH-tibolone (n = 3). In conclusion, SULT2A1 is the major endogenous enzyme responsible for sulfation of the tibolone metabolites in human postmenopausal tissues. The results support the occurrence of pre-receptor enzymatic regulation of hydroxytibolone metabolites and prompt further investigation of the tissue-selective regulation of tibolone effects.     

Aetiology of skeletal deformities in the Zebra Danio fish (Bruchydanio rerio, Hamilton-Buchanan)

Laboratory bred Zebra Danio ( Brachydanio rerio , Hamilton-Buchanan) tropical fish are prone to skeletal deformities resembling scoliosis and lordosis. This condition appears related to diet and has been studied by breeding from different broods of fish for three generations. Two broods bred using two commercially available tropical flaked foods developed severe spinal curvature three to six weeks post-hatching. A third brood of fish fed exclusively on live food did not develop any deformity. The results demonstrate that a dietary factor is responsible for the deformities and that the Zebra Danio is especially susceptible to this factor as other species breed normally when fed the same commercial diet. Analysis of the diets showed no deficiency in either ascorbic acid (Vitamin C) or tryptophan, deficiency of either being associated with scoliosis and lordosis in fish. The analysis did show, however, a relationship between the lead content of the diet and the incidence of deformities. Lead has previously been implicated in skeletal deformities in second and third generation Brook trout, Salvelinus fontinalis.     

Spontaneous skeletal deformities in the Zebra Danio (Brachydanio rerio) bred for fish toxicity tests

Zebra Danio ( Brachydanio rerio , Hamilton-Buchanan) tropical fish are easily maintained and bred under laboratory conditions; procedures for maintenance and breeding are described. Intensive inbreeding of this fish gave rise to a high incidence of spontaneous skeletal deformities which appear to reflect a genetic defect rather than an environmental stimulus. This finding casts doubt on the suitability of the Zebra Danio for fish toxicity testing.     

Developmental expression of alcohol dehydrogenase (ADH3) in zebrafish (Danio rerio)

Alcohol dehydrogenase (ADH) is the primary enzyme responsible for metabolism of ethanol to acetaldehyde. One class of ADH has been described in fish, and has been found to be structurally similar to mammalian class III ADH (glutathione-dependent formaldehyde dehydrogenase) but functionally similar to class I ADH (primarily responsible for ethanol metabolism). We have cloned a cDNA by RT-PCR from zebrafish (Danio rerio) liver representing the zebrafish ADH3 gene product, with a coding region of 1131 nucleotides. The deduced amino acid sequences share 90% identity to ADH3 from the marine fish Sparus aurata, and 82 and 81% identity to the mouse and human sequences, respectively. Using a quantitative competitive RT-PCR assay, ADH3 mRNA was detected at all timepoints analyzed and was lowest between 8 and 24 h postfertilization. Thus, differential ADH3 expression may be at least partly responsible for temporal variations in the sensitivity of zebrafish embryos to developmental alcohol exposure.     

Sulfation of ractopamine and salbutamol by the human cytosolic sulfotransferases

Feed additives such as ractopamine and salbutamol are pharmacologically active compounds, acting primarily as β-adrenergic agonists. This study was designed to investigate whether the sulfation of ractopamine and salbutamol may occur under the metabolic conditions and to identify the human cytosolic sulfotransferases (SULTs) that are capable of sulfating two major feed additive compounds, ractopamine and salbutamol. A metabolic labelling study showed the generation and release of [(35)S]sulfated ractopamine and salbutamol by HepG2 human hepatoma cells labelled with [(35)S]sulfate in the presence of these two compounds. A systematic analysis using 11 purified human SULTs revealed SULT1A3 as the major SULT responsible for the sulfation of ractopamine and salbutamol. The pH dependence and kinetic parameters were analyzed. Moreover, the inhibitory effects of ractopamine and salbutamol on SULT1A3-mediated dopamine sulfation were investigated. Cytosol or S9 fractions of human lung, liver, kidney and small intestine were examined to verify the presence of ractopamine-/salbutamol-sulfating activity in vivo. Of the four human organs, the small intestine displayed the highest activity towards both compounds. Collectively, these results imply that the sulfation mediated by SULT1A3 may play an important role in the metabolism and detoxification of ractopamine and salbutamol.     

Ammonia excretion by the skin of zebrafish (Danio rerio) larvae

The mechanism of ammonia excretion in freshwater teleosts is not well understood. In this study, scanning ion-selective electrode technique was applied to measure H(+) and NH(4)(+) fluxes in specific cells on the skin of zebrafish larvae. NH(4)(+) extrusion was relatively high in H(+) pump-rich cells, which were identified as the H(+)-secreting ionocyte in zebrafish. Minor NH(4)(+) extrusion was also detected in keratinocytes and other types of ionocytes in larval skin. NH(4)(+) extrusion from the skin was tightly linked to acid secretion. Increases in the external pH and buffer concentration (5 mM MOPS) diminished H(+) and NH(4)(+) gradients at the larval surface. Moreover, coupled decreases in NH(4)(+) and H(+) extrusion were found in larvae treated with an H(+)-pump inhibitor (bafilomycin A1) or H(+)-pump gene (atp6v1a) knockdown. Knockdown of Rhcg1 with morpholino-oligonucleotides also decreased NH(4)(+) excretion. This study demonstrates ammonia excretion in epithelial cells of larval skin through an acid-trapping mechanism, and it provides direct evidence for the involvement of the H(+) pump and an Rh glycoprotein (Rhcg1) in ammonia excretion.     

The Influence of Infective Dose on the Virulence of a Generalist Pathogen in Rainbow Trout (Oncorhynchus mykiss) and Zebra Fish (Danio rerio)

Pathogen density and genetic diversity fluctuate in the outside-host environment during and between epidemics, affecting disease emergence and the severity and probability of infections. Although the importance of these factors for pathogen virulence and infection probability has been acknowledged, their interactive effects are not well understood. We studied how an infective dose in an environmentally transmitted opportunistic fish pathogen, Flavobacterium columnare, affects its virulence both in rainbow trout, which are frequently infected at fish farms, and in zebra fish, a host that is not naturally infected by F. columnare. We used previously isolated strains of confirmed high and low virulence in a single infection and in a co-infection. Infection success (measured as host morbidity) correlated positively with dose when the hosts were exposed to the high-virulence strain, but no response for the dose increase was found when the hosts were exposed to the low-virulence strain. Interestingly, the co-infection resulted in poorer infection success than the single infection with the high-virulence strain. The rainbow trout were more susceptible to the infection than the zebra fish but, in both species, the effects of the doses and the strains were qualitatively similar. We suggest that as an increase in dose can lead to increased host morbidity, both the interstrain interactions and differences in infectivity in different hosts may influence the severity and consequently the evolution of disease. Our results also confirm that the zebra fish is a good laboratory model to study F. columnare infection.     

Using transgenic zebrafish (Danio rerio) to study development of the craniofacial skeleton

Transgenic zebrafish provide amazing new tools for following and manipulating cells that form the skeleton. Transgenes that label distinct populations of embryonic cells, such as neural crest that forms most of the skull vault as well as the jaws and gills can be used to determine the embryonic origins of cartilages and bones. This provides a powerful model system for studies of the developmental basis for human birth defects and in a comparative context provides new insights into the developmental changes underlying morphological evolution. Targeting transgenes to nuclear or membrane compartments allows detailed tracking of cell shapes and movements. Here we review how such transgenic markers combined with mutants or tissue grafts to generate mosaic zebrafish embryos have already provided many new insights into skeletal development and disease. In the long run, transgenics designed to perturb gene expression hold great promise for studies of gene function.     

Sulfation of tibolone and tibolone metabolites by expressed human cytosolic sulfotransferases

Tibolone is an important therapeutic agent used in the treatment of menopausal symptoms in many countries and has beneficial effects on menopausal and postmenopausal vasomotor, bone, vaginal and mood symptoms without affecting the endometrial, breast or cardiovascular systems. The rapid metabolism of tibolone to active metabolites including 3-OH-tibolone, 3β-OH-tibolone and Δ⁴-tibolone may be important in its tissue-specific effects. Sulfation also has a major role in the metabolism and regulation of the tissue-specific activity of tibolone and its metabolites. The ability of seven major expressed human sulfotransferase (SULT) isoforms to sulfate tibolone and its three metabolites was examined. Expressed human SULT2A1 was capable of sulfating tibolone and all three metabolites with the highest affinity for 3-OH-tibolone. SULT1E1 conjugated both 3-OH-tibolone metabolites and tibolone itself slightly. SULT2B1b sulfated both 3-OH metabolites but not tibolone or Δ⁴-tibolone. SULT isoforms 1A1, 1A3, 1B1 and 1C1 did not demonstrate detectable activity. Sulfation of tibolone and its metabolites by human tissue cytosols was analyzed to determine whether the pattern of tibolone sulfation corresponded to the known expression of SULT isoforms in each tissue. The tissue-specific effects of tibolone may be regulated in part by the inactivation of tibolone and its metabolites by specific human SULT isoforms.     

Identification of a puromycin-sensitive aminopeptidase from zebrafish (Danio rerio)

An aminopeptidase from zebrafish (Danio rerio) was purified 1247-fold to homogeneity with 35.4% recovery by column chromatography successively on DEAE-sephacel, hydroxyapatite, and phenyl-sepharose. The molecular mass of the enzyme was estimated at 98 kDa by SDS-PAGE and gel filtration. Optimum temperature and pH of the enzyme were 45°C and 7.5, respectively. The enzyme preferentially hydrolyzed substrate Leu-MCA with k(cat)/K(m) of 4.2×10(6)M(-1)s(-1) and an activation energy of 68.9 kJ M(-1) [corrected], respectively. It was specifically inhibited by bestatin, puromycin and metal-chelating agents, and Zn(2+) seemed to be its metal cofactor(s). Some l-amino acids significantly inhibited its activity, and l-cysteine was a non-competitive inhibitor with a K(i) of 0.27 mM. According to the peptide mass fingerprint analysis, the enzyme was highly matched with the predicted D. rerio aminopeptidase puromycin sensitive (gi: 255683530) (EC 3.4.11.14), suggesting that the present enzyme is a puromycin-sensitive aminopeptidase of zebrafish.     

Sulfation of flavonoids and other phenolic dietary compounds by the human cytosolic sulfotransferases

The protective effects of diet, especially soya products, tea, and many fruits, against a variety of human cancers, as suggested by epidemiological studies, has focused attention on flavonoids, isoflavonoids, and other phenolic dietary compounds as chemoprotectants. Among the mechanisms suggested for their chemoprotective action, their ability to inhibit the bioactivation of carcinogens by the human cytosolic sulfotransferases (STs) and the direct effects of their sulfoconjugates are being increasingly studied. We report here a systematic study on the sulfation of representative flavonoids, isoflavonoids, anti-oxidants, and other phenolic dietary compounds by all ten known human cytosolic STs. All ten recombinant human cytosolic STs were prepared in a pure form and tested for their sulfating activities with a variety of these compounds. P-form (SULT1A1) phenol ST (PST) showed high sulfating activity with most of these compounds. M-form (SULT1A3) PST showed high activity with the flavonoids but not with the isoflavonoids. SULT1C ST #2 showed high activity with the isoflavonoids and also sulfated most of the other compounds. Possible relevance of these results to the chemoprotective effects of these dietary compounds is discussed.     

The use of mature zebrafish (Danio rerio) as a model for human aging and disease

Zebrafish (Danio rerio) have been extensively utilized for understanding mechanisms of development. These studies have led to a wealth of resources including genetic tools, informational databases, and husbandry methods. In spite of all these resources, zebrafish have been underutilized for exploring pathophysiology of disease and the aging process. Zebrafish offer several advantages over mammalian models for these studies, including the ability to perform saturation mutagenesis and the capability to contain thousands of animals in a small space. In this review, we will discuss the use of mature zebrafish as an animal model and provide specific examples to support this novel use of zebrafish. Examples include demonstrating that clinical pathology can be performed in mature zebrafish and that age-associated changes in heat shock response can be observed in zebrafish. These highlights demonstrate the utility of zebrafish as a model for disease and aging.     

Proteomic analysis of zebrafish (Danio rerio) embryos exposed to cyclosporine A

Cyclosporine A, a potent immunosuppressive agent extensively used to prevent allograft rejections, is under scrutiny due to severe toxic effects. CsA therapy is often continued during pregnancy in conditions such as organ transplantations and autoimmune diseases. Herein, we investigated the effects of CsA on early morphogenesis of zebrafish and identified a spectrum of proteins whose expression was altered in the drug treated embryos. Time-lapse fluorescence imaging of germ-line double transgenic zebrafish embryos treated with CsA revealed severe blood regurgitation in heart chambers, absence of blood circulation in vessels, pericardial and yolk sac edema. We also observed lack of mature blood vessels and down-regulation of endothelial markers in CsA treated embryos. Proteomic analysis using 2D-DIGE followed by mass-spectrometry led to the identification of 37 proteins whose expression was significantly modulated in presence of the drug. These proteins were mostly associated with cytoskeletal/structural assembly, lipid-binding, stress response and metabolism. Furthermore, mRNA expression analysis of eight proteins and Western blotting of actin revealed consistency between the changes observed in protein expression and its corresponding mRNA levels. Our findings demonstrate that CsA administration during early morphogenesis in zebrafish modulates the expression of some proteins which are known to be involved in important physiological processes.     

A single amino acid mutation in zebrafish (Danio rerio) liver bile acid-binding protein can change the stoichiometry of ligand binding

In all of the liver bile acid-binding proteins (L-BABPs) studied so far, it has been found that the stoichiometry of binding is of two cholate molecules per internal binding site. In this paper, we describe the expression, purification, crystallization, and three-dimensional structure determination of zebrafish (Danio rerio) L-BABP to 1.5A resolution, which is currently the highest available for a protein of this family. Since we have found that in zebrafish, the stoichiometry of binding in the protein cavity is of only one cholate molecule per wild type L-BABP, we examined the role of two crucial amino acids present in the binding site. Using site-directed mutagenesis, we have prepared, crystallized, and determined the three-dimensional structure of co-crystals of two mutants. The mutant G55R has the same stoichiometry of binding as the wild type protein, whereas the C91T mutant changes the stoichiometry of binding from one to two ligand molecules in the cavity and therefore appears to be more similar to the other members of the L-BABP family. Based on the presence or absence of a single disulfide bridge, it can be postulated that fish should bind a single cholate molecule, whereas amphibians and higher vertebrates should bind two. Isothermal titration calorimetry has also revealed the presence in the wild type protein and the G55R mutant of an additional binding site, different from the first and probably located on the surface of the molecule.