Hybrid Allosteric Modulators of M1 Muscarinic Receptors Enhance Acetylcholine Efficacy and Decrease Locomotor Activity and Turning Behaviors in Zebrafish.

Allosteric modulation of muscarinic acetylcholine receptors (mAChR) has been identified as a potential strategy for regulating cholinergic signaling in the treatment of various neurological disorders. Most positive allosteric modulators (PAMs) of mAChR enhance agonist affinity and potency, while very few PAMs selectively enhance G-protein coupling efficacy (e.g., amiodarone). The key structural features of amiodarone responsible for enhancement of mAChR efficacy were examined in CHO cells expressing M1 receptors. Subsequent incorporation of these structural features into previously identified allosteric modulators of potency (i.e., n-benzyl isatins) generated hybrid ligands that demonstrated similar or better enhancement of mAChR efficacy, lower in vivo toxicity, and higher allosteric binding affinity relative to amiodarone. Notable hybrid ligands include 8a and 8b which respectively demonstrated the strongest binding affinity and the most robust enhancement of mAChR efficacy as calculated from an allosteric operational model. Amiodarone derivatives and hybrid ligands were additionally screened in wildtype zebrafish (Danio rerio) to provide preliminary in vivo toxicity data as well as to observe effects on locomotor and turning behaviors relative to other mAChR PAMs. Several compounds, including 8a and 8c, reduced locomotor activity and increased measures of turning behaviors in zebrafish, suggesting that allosteric modulation of muscarinic receptor efficacy might be useful in the treatment of repetitive behaviors associated with autism spectrum disorder (ASD) and other neuropsychiatric disorders.

Development of Liquid Chromatography-Tandem Mass Spectrometry Analytical Methods for the Study of Whole-Body, Head, and Trunk Uptake and Elimination of Methamphetamine in 5-Day Postfertilization Zebrafish Larvae Using the Quick, Easy, Cheap, Effective, Rugged, and Safe Technique.

Synthetic cathinones are drugs of abuse substituted for amphetamine-like stimulant drugs such as methamphetamine. In this study, methamphetamine was studied as a prototypical amphetamine-like drug as a first step toward establishing methods to study this entire drug class. The internal concentration of methamphetamine in zebrafish larvae was determined using matrix-matched calibration along with extraction and purification of samples using the quick, easy, cheap, effective, rugged, and safe technique in liquid chromatography-tandem mass spectrometry. Whole-body and head/trunk uptake and elimination in 5-day postfertilization zebrafish larvae were determined. A gradient method was developed using 5 mM ammonium formate with 0.1% formic acid and methanol with 0.1% formic acid as mobile phases, 10 min of total run time, and a 0.3 mL/min flow rate. The limit of quantification was 60 ng/mL, linearity with r2 = 0.9991, and recovery values from 92% to 120%. The internal concentration of methamphetamine was quantifiable in whole-body homogenates within 15 min of uptake analysis. The internal concentration increased with time, whereas a biphasic elimination pattern was shown. With increasing length of exposure, a higher accumulation of drugs was found in the head than in the trunk.

Methamphetamine-induced lethal toxicity in zebrafish larvae.

RATIONALE: The use of novel psychoactive substances has been steadily increasing in recent years. Given the rapid emergence of new substances and their constantly changing chemical structure, it is necessary to develop an efficient and expeditious approach to examine the mechanisms underlying their pharmacological and toxicological effects. Zebrafish (Danio rerio) have become a popular experimental subject for drug screening due to their amenability to high-throughput approaches. OBJECTIVES: In this study, we used methamphetamine (METH) as an exemplary psychoactive substance to investigate its acute toxicity and possible underlying mechanisms in 5-day post-fertilization (5 dpf) zebrafish larvae. METHODS: Lethality and toxicity of different concentrations of METH were examined in 5-dpf zebrafish larvae using a 96-well plate format. RESULTS: METH induced lethality in zebrafish larvae in a dose-dependent manner, which was associated with initial sympathomimetic activation, followed by cardiotoxicity. This was evidenced by significant heart rate increases at low doses, followed by decreased cardiac function at high doses and later time points. Levels of ammonia in the excreted water were increased but decreased internally. There was also evidence of seizures. Co-administration of the glutamate AMPA receptor antagonist GYKI-52466 and the dopamine D2 receptor antagonist raclopride significantly attenuated METH-induced lethality, suggesting that this lethality may be mediated synergistically or independently by glutamatergic and dopaminergic systems. CONCLUSIONS: These experiments provide a baseline for the study of the toxicity of related amphetamine compounds in 5-dpf zebrafish as well as a new high-throughput approach for investigating the toxicities of rapidly emerging new psychoactive substances.

Combining Neurobehavioral Analysis and In Vivo Photoaffinity Labeling to Understand Protein Targets of Methamphetamine in Casper Zebrafish.

Photoaffinity labeling (PAL) remains one of the most widely utilized methods of determining protein targets of drugs. Although useful, the scope of this technique has been limited to in vitro applications because of the inability of UV light to penetrate whole organisms. Herein, pigment-free Casper zebrafish were employed to allow in vivo PAL. A methamphetamine-related phenethylamine PAL probe, designated here as 2, demonstrated dose-dependent effects on behavior similar to methamphetamine and permitted concentration-dependent labeling of protein binding partners. Click chemistry was used to analyze binding partners via fluoroimaging. Conjugation to a biotin permitted streptavidin pull-down and proteomic analysis to define direct binding partners of the methamphetamine probe. Bioinformatic analysis revealed the probe was chiefly bound to proteins involved in phagocytosis and mitochondrial function. Future applications of this experimental paradigm combining examination of drug-protein binding interactions alongside neurobehavioral readouts via in vivo PAL will significantly enhance our understanding of drug targets, mechanism(s) of action, and toxicity/lethality.

Novel Thienopyrimidine Derivative, RP-010, Induces ß-Catenin Fragmentation and Is Efficacious against Prostate Cancer Cells.

Thienopyrimidines containing a thiophene ring fused to pyrimidine are reported to have a wide-spectrum of anticancer efficacy in vitro. Here, we report for the first time that thieno[3,2-d]pyrimidine-based compounds, also known as the RP series, have efficacy in prostate cancer cells. The compound RP-010 was efficacious against both PC-3 and DU145 prostate cancer (PC) cells (IC50 < 1 µM). The cytotoxicity of RP-010 was significantly lower in non-PC, CHO, and CRL-1459 cell lines. RP-010 (0.5, 1, 2, and 4 µM) arrested prostate cancer cells in G2 phase of the cell cycle, and induced mitotic catastrophe and apoptosis in both PC cell lines. Mechanistic studies suggested that RP-010 (1 and 2 µM) affected the wingless-type MMTV (Wnt)/ß-catenin signaling pathway, in association with ß-catenin fragmentation, while also downregulating important proteins in the pathway, including LRP-6, DVL3, and c-Myc. Interestingly, RP-010 (1 and 2 µM) induced nuclear translocation of the negative feedback proteins, Naked 1 and Naked 2, in the Wnt pathway. In addition, RP-010 (0.5, 1, 2 and 4 µM) significantly decreased the migration of PC cells in vitro. Finally, RP-010 did not produce significant toxic effects in zebrafish at concentrations of up to 6 µM. In conclusion, RP-010 may be an efficacious and relatively nontoxic anticancer compound for prostate cancer. Future mechanistic and in vivo efficacy studies are needed to optimize the hit compound RP-010 for lead optimization and clinical use.

Developmental effects of trichloroacetate in Zebrafish embryos: Association with the production of superoxide anion.

To assess the developmental toxicity of trichloroacetate (TCA), zebrafish embryos were exposed to 8 to 48 mM of TCA and evaluated for developmental milestones from 8- to 144-hour postfertilization (hpf). All developmental toxicities are reported in this paper. Embryos were found to have developed edema in response to 16 to 48 mM of TCA exposure at 32- to 80-hpf, experienced delay in hatching success in response to 24 to 48 mM at 80-hpf. Lordosis was observed in developing embryos exposed to 40 to 48 mM at 55- to 144-hpf. The observed toxic effects of TCA exposure were found to be concentration and exposure period independent. Effects were found to be associated with increases in superoxide anion production, but these increases were also found to be concentration and time independent. TCA resulted in concentration-dependent increases in embryonic lethality at 144-hpf, with an LC50 determined to be 29.7 mM.

Identification of zebrafish steroid sulfatase and comparative analysis of the enzymatic properties with human steroid sulfatase.

Steroid sulfatase (STS) plays an important role in the regulation of steroid hormones. Metabolism of steroid hormones in zebrafish has been investigated, but the action of steroid sulfatase remains unknown. In this study, a zebrafish sts was cloned, expressed, purified, and characterized in comparison with the orthologous human enzyme. Enzymatic assays demonstrated that similar to human STS, zebrafish Sts was most active in catalyzing the hydrolysis of estrone-sulfate and estradiol-sulfate, among five steroid sulfates tested as substrates. Kinetic analyses revealed that the Km values of zebrafish Sts and human STS differed with respective substrates, but the catalytic efficiency as reflected by the Vmax/Km appeared comparable, except for DHEA-sulfate with which zebrafish Sts appeared less efficient. While zebrafish Sts was catalytically active at 28 °C, the enzyme appeared more active at 37 °C and with similar Km values to those determined at 28 °C. Assays performed in the presence of different divalent cations showed that the activities of both zebrafish and human STSs were stimulated by Ca2+, Mg2+, and Mn2+, and inhibited by Zn+2 and Fe2+. EMATE and STX64, two known mammalian steroid sulafatase inhibitors, were shown to be capable of inhibiting the activity of zebrafish Sts. Collectively, the results obtained indicated that zebrafish Sts exhibited enzymatic characteristics comparable to the human STS, suggesting that the physiological function of STS may be conserved between zebrafish and humans.

The GSK3ß inhibitor, TDZD-8, rescues cognition in a zebrafish model of okadaic acid-induced Alzheimer's disease.

Currently, no treatments exist that are able to directly treat against Alzheimer's disease (AD), and we are facing an inevitable increase in the near future of the amount of patients who will suffer from AD. Most animal models of AD are limited by not being able to recapitulate the entire pathology of AD. Recently an AD model in zebrafish was established by using the protein phosphatase 2A inhibitor, okadaic acid (OKA). Administering OKA to zebrafish was able to recapitulate most of the neuropathology associated with AD. Therefore, providing a drug discovery model for AD that is also time and cost efficient. This study was designed to investigate the effects of GSK3ß inhibition by 4-benzyl-2-methyl-1, 2, 4-thiadiazolidine-3, 5-dione (TDZD-8) on this newly developed AD model. Fish were divided into 4 groups and each group received a different treatment. The fish were divided into a control group, a group treated with 1 µM TDZD-8 only, a group treated with 1 µM TDZD-8 + 100 nM OKA, and a group treated with 100 nM OKA only. Administering the GSK3ß inhibitor to zebrafish concomitantly with OKA proved to be protective. TDZD-8 treatment reduced the mortality rate, the ratio of active: inactive GSK3ß, pTau (Ser199), and restored PP2A activity. This further corroborates the use of GSKß inhibitors in the treatment against AD and bolsters the use of the OKA-induced AD-like zebrafish model for drug discovery.

Utilizing zebrafish and okadaic acid to study Alzheimer's disease.

Despite the many years of extensive research using rodent models to study Alzheimer's disease (AD), no cure or disease halting drug exists. An increasing number of people are suffering from the disease and a therapeutic intervention is needed. Therefore, it is necessary to have complementary models to aid in the drug discovery. The zebrafish animal model is emerging as a valuable model for the investigation of AD and neurodegenerative drug discovery. The main genes involved in human AD have homologous counterparts in zebrafish and have conserved function. The basic brain structure of the zebrafish is also conserved when compared to the mammalian brain. Recently an AD model was established by administering okadaic acid to zebrafish. It was used to test the efficacy of a novel drug, lanthionine ketimine-5-ethyl ester, and to elucidate its mechanism of action. This demonstrated the ability of the okadaic acid-induced AD zebrafish model to be implemented in the drug discovery process for therapeutics against AD.

Differential Toxicity of mDia Formin-Directed Functional Agonists and Antagonists in Developing Zebrafish.

The mammalian Diaphanous-related (mDia) formins are cytoskeletal regulators that assemble and, in some cases, bundle filamentous actin (F-actin), as well as stabilize microtubules. The development of small molecule antagonists and agonists that interrogate mDia formin function has allowed us to investigate the roles of formins in disease states. A small molecule inhibitor of FH2 domain (SMIFH2) inhibits mDia-dependent actin dynamics and abrogates tumor cell migration and cell division in vitro and ex vivo tissue explants. mDia formin activation with small molecule intramimics IMM01/02 and mDia2-DAD peptides inhibited glioblastoma motility and invasion in vitro and ex vivo rat brain slices. However, SMIFH2, IMMs, and mDia2 DAD efficacy in vivo remains largely unexplored and potential toxicity across a range of developmental phenotypes has not been thoroughly characterized. In this study, we performed an in vivo screen of early life-stage toxicity in Danio rerio zebrafish embryos 2 days post-fertilization (dpf) in response to SMIFH2, IMM01/02, and mDia2 DAD. SMIFH2 at concentrations ≥5-10 µM induced significant defects in developing zebrafish, including shorter body lengths, tail curvature and defective tail cellularity, craniofacial malformations, pericardial edema, absent and/or compromised vasculature function and flow, depressed heart rates and increased mortality. Conversely, IMM and mDia2 DAD peptides were minimally toxic at concentrations up to 10-20 and 50 µM, respectively. SMIFH2's therapeutic potential may therefore be limited by its substantial in vivo toxicity at functional concentrations. mDia formin agonism with IMMs and mDia2 DADs may therefore be a more effective and less toxic anti-invasive therapeutic approach.

Lanthionine ketimine-5-ethyl ester provides neuroprotection in a zebrafish model of okadaic acid-induced Alzheimer's disease.

Okadaic acid (OKA) is a protein phosphatase 2A inhibitor that is used to induce neurodegeneration and study disease states such as Alzheimer's disease (AD). Lanthionine ketimine-5-ethyl ester (LKE) is a bioavailable derivative of the naturally occurring brain sulfur metabolite, lanthionine ketimine (LK). In previously conducted studies, LKE exhibited neuroprotective and neurotrophic properties in murine models but its mechanism of action remains to be clarified. In this study, a recently established zebrafish OKA-induced AD model was utilized to further elucidate the neuroprotective and neurotrophic properties of LKE in the context of an AD-like condition. The fish were divided into 3 groups containing 8 fish per group. Group #1 = negative control, Group #2 = 100 nM OKA, Group #3 = 100 nM OKA +500 µM LKE. OKA caused severe cognitive impairments in the zebrafish, but concomitant treatment with LKE protected against cognitive impairments. Further, LKE significantly and substantially reduced the number of apoptotic brain cells, increased brain-derived neurotrophic factor (BDNF), and increased phospho-activation of the pro-survival factors pAkt (Ser 473) and pCREB (Ser133). These findings clarify the neuroprotective and neurotrophic effects of LKE by highlighting particular survival pathways that are bolstered by the experimental therapeutic LKE.

Identification and characterization of 5α-cyprinol-sulfating cytosolic sulfotransferases (Sults) in the zebrafish (Danio rerio).

5α-Cyprinol 27-sulfate is the major biliary bile salt present in cypriniform fish including the zebrafish (Danio rerio). The current study was designed to identify the zebrafish cytosolic sulfotransferase (Sult) enzyme(s) capable of sulfating 5α-cyprinol and to characterize the zebrafish 5α-cyprinol-sulfating Sults in comparison with human SULT2A1. Enzymatic assays using zebrafish homogenates showed 5α-cyprinol-sulfating activity. A systematic analysis, using a panel of recombinant zebrafish Sults, revealed two Sult2 subfamily members, Sult2st2 and Sult2st3, as major 5α-cyprinol-sulfating Sults. Both enzymes showed higher activities using 5α-cyprinol as the substrate, compared to their activity with DHEA, a representative substrate for mammalian SULT2 family members, particularly SULT2A1. pH-Dependence and kinetics experiments indicated that the catalytic properties of zebrafish Sult2 family members in mediating the sulfation of 5α-cyprinol were different from those of either zebrafish Sult3st4 or human SULT2A1. Collectively, these results imply that both Sult2st2 and Sult2st3 have evolved to sulfate specifically C27-bile alcohol, 5α-cyprinol, in Cypriniform fish, whereas the enzymatic characteristics of zebrafish Sult3 members, particularly Sult3st4, correlated with those of human SULT2A1.

HM015k, a Novel Silybin Derivative, Multi-Targets Metastatic Ovarian Cancer Cells and Is Safe in Zebrafish Toxicity Studies.

This study was designed to determine the in vitro mechanisms by which the novel silybin derivative, (E)-3-(3-(benzyloxy) phenyl)-1-(4-hydroxyphenyl)prop-2-en-1-one (HM015k or 15k), produces its anticancer efficacy in ovarian cancer cells. Compound 15k induced apoptosis in ovarian cancer cells in a time-dependent manner by significantly upregulating the expression of Bax and Bak and downregulating the expression of Bcl-2. Interestingly, 15k induced the cleavage of Bax p21 into its more efficacious cleaved form, Bax p18. In addition, caspase 3 and caspase 9 were cleaved to their active forms, inducing the cleavage of poly ADP ribose polymerase (PARP) and ß-catenin. Furthermore, in OV2008 cells, 15k induced significant cleavage in nuclear ß-catenin to primarily inactive fragments of lower molecular weight. Furthermore, 15k reversed the metastatic potential of OV2008 cells by inhibiting their migration and invasiveness. The mesenchymal phenotype in OV2008 was reversed by 15k, causing cells to be rounder with epithelial-like phenotypes. The 15k-induced reversal was further confirmed by significant upregulation of the E-cadherin expression, an epithelial marker, while N-cadherin, a mesenchymal marker, was downregulated in OV2008 cells. Compound 15k inhibited the expression of the oncogenic c-Myc protein, downregulated proteins DVL3 and DVL2 and significantly upregulated cyclin B1. Also, 15k significantly downregulated the expression levels of ABCG2 and ABCB1 transporters in resistant ABCG2 overexpressing H460/MX20 and resistant ABCB1 overexpressing MDCK/MDR1 cells, respectively. Finally, 15k was safe in zebrafish in vivo model at concentrations up to 10 µM and induced no major toxicities in cardiac, morphology and swimming position parameters. Overall, 15k is a multi-targeted inhibitor with efficacy against metastatic and resistant ovarian cancer. Future in vivo studies will be conducted to determine the efficacy of 15k in tumor-bearing animals.

Development of a Novel and Robust Pharmacological Model of Okadaic Acid-induced Alzheimer's Disease in Zebrafish.

Alzheimer's disease (AD) is the leading neurodegenerative disorder affecting the world's elderly population. Most experimental models of AD are transgenic or pharmacological in nature, and do not simulate the entire pathophysiology. In the present study, we developed a pharmacologically induced AD using the zebrafish, a species that can recapitulate most of the phenotypes of the disease. The pharmacological agent being used, okadaic acid (OKA) has also been utilized to study AD in other species. In this model, the immunohistochemistry of phosphorylated glycogen synthase-3α/ß, Aß, p-tau, tau protein, and senile plaque formation in zebrafish brain were all significantly increased with increasing exposure to OKA. These represent the majority of the histological hallmarks of AD pathophysiology. The observed changes were also accompanied by learning and memory deficits which are also important components in AD pathophysiology. Zebrafish disease models are gaining popularity mostly due to their economic cost and relevance to human disease pathophysiology. Current pharmacological methods of inducing AD in zebrafish are not adequately developed and do not represent all the features of the disease. OKA-induced AD in zebrafish can become a cost efficient model to study drug discovery for AD. It may also be used to unravel the molecular mechanisms underlying the complex pathophysiology that leads to AD using relatively economical species.

Sulfation of benzyl alcohol by the human cytosolic sulfotransferases (SULTs): a systematic analysis.

The aim of the present study was to identify human cytosolic sulfotransferases (SULTs) that are capable of sulfating benzyl alcohol and to examine whether benzyl alcohol sulfation may occur in cultured human cells as well as in human organ homogenates. A systematic analysis revealed that of the 13 known human SULTs, SULT1A1 SULT1A2, SULTA3, and SULT1B1 are capable of mediating the sulfation of benzyl alcohol. The kinetic parameters of SULT1A1 that showed the strongest benzyl alcohol-sulfating activity were determined. HepG2 human hepatoma cells were used to demonstrate the generation and release of sulfated benzyl alcohol under the metabolic settings. Moreover, the cytosol or S9 fractions of human liver, lung, kidney and small intestine were examined to verify the presence of benzyl alcohol sulfating activity in vivo. Copyright © 2015 John Wiley & Sons, Ltd.

Primary cilium regulates CaV1.2 expression through Wnt signaling.

Primary cilia are sensory organelles that provide a feedback mechanism to restrict Wnt signaling in the absence of endogenous Wnt activators. Abnormal Wnt signaling has been shown to result in polycystic kidney disease (PKD) although the exact mechanism has been debated. Previously, we reported that the calcium channel CaV1.2 functions in primary cilia. In this study, we show that CaV1.2 expression level is regulated by Wnt signaling. This occurs through modulation of mitochondrial mass and activity resulting in increased reactive oxygen species which generate oxidative DNA lesions. We found that the subsequent cellular DNA damage response triggers increased CaV1.2 expression. In the absence of primary cilia where Wnt signaling is upregulated, we found that CaV1.2 is overexpressed as a compensatory mechanism. We show for the first time that CaV1.2 knockdown in zebrafish results in classic primary cilia defects including renal cyst formation, hydrocephalus, and left-right asymmetry defects. Our study shows that suppressed Wnt signaling prevents CaV1.2 expression ultimately resulting in PKD phenotypes. Thus, CaV1.2 expression is tightly regulated through Wnt signaling and plays an essential sensory role in primary cilia necessary for cellular homeostasis.

Development and characterization of nanostructured mists with potential for actively targeting poorly water-soluble compounds into the lungs.

PURPOSE: To formulate nanoemulsions (NE) with potential for delivering poorly water-soluble drugs to the lungs. METHOD: A self nanoemulsifying composition consisting of cremophor RH 40, PEG 400 and labrafil M 2125 CS was selected after screening potential excipients. The solubility of carbamazepine, a poorly water-soluble drug, was tested in the formulation components. Oil-in-water (o/w) NEs were characterized using dynamic light scattering, electrophoretic light scattering, transmission electron microscopy (TEM) and differential scanning calorimetry. NEs were nebulized into a mist using a commercial nebulizer and characterized using laser diffraction and TEM. An aseptic method was developed for preparing sterile NEs. Biocompatibility of the formulation was evaluated on NIH3T3 cells using MTT assay. In vitro permeability of the formulation was tested in zebra fish eggs, HeLa cells, and porcine lung tissue. RESULTS: NEs had neutrally charged droplets of less than 20 nm size. Nebulized NEs demonstrated an o/w nanostructure. The mist droplets were of size less than 5 µm. Sterility testing and cytotoxicity results validated that the NE was biocompatible and sterile. In vitro tests indicated oil nanodroplets penetrating intracellularly through biological membranes. CONCLUSION: The nanoemulsion mist has the potential for use as a pulmonary delivery system for poorly water-soluble drugs.

Preparation, characterization, sterility validation, and in vitro cell toxicity studies of microemulsions possessing potential parenteral applications.

CONTEXT: Water-in-oil microemulsions (w/o ME) are ideal for parenteral drug delivery. However, no such formulations have been tested for biocompatibility in in vitro cell cultures. Furthermore, sterilization of w/o MEs is a challenging process that has not been previously developed and validated. PURPOSE: To formulate pharmaceutically relevant water-in-oil (w/o) microemulsion's systems suitable for use as a parenteral formulation. METHODS: w/o MEs were prepared using dioctyl sodium sulfosuccinate (DOSS), ethyl oleate (EO), and water. Formulations were characterized using polarized light microscopy, electrical conductivity, rheology, and dynamic light scattering. An aseptic filtration method for sterilization was developed using membrane filtration. The biocompatibility of selected MEs were evaluated in NIH3T3 cell cultures. Dissolution studies were performed on microemulsions containing methylene blue to evaluate the drug release profile. RESULTS: The maximum amount of water incorporated in the formulations was 14% w/w. DOSS/EO/water microemulsions exhibited Newtonian flow. Particle sizes for these MEs were less than 30 nm in size. Formulations filtered aseptically were free of bacteria when gram-stained and visualized under a microscope. All MEs showed no toxicity to NIH 3T3 cells. DISCUSSION: The absence of birefringence and low conductivity values indicated that the formulations were w/o microemulsions. The filtration method of sterilization was validated by the absence of microbial growth on blood agar plates over a 14-day period. In vitro dye release studies demonstrate sustained release of the model drug over a 72-h time period. CONCLUSION: Characteristics delineated in this study demonstrate the potential for these formulations to be used as parenteral preparations.

Identification and characterization of zebrafish SULT1 ST9, SULT3 ST4, and SULT3 ST5.

By searching the GenBank database, we identified sequences encoding three new zebrafish cytosolic sulfotransferases (SULTs). These three new zebrafish SULTs, designated SULT1 ST9, SULT3 ST4, and SULT3 ST5, were cloned, expressed, purified, and characterized. SULT1 ST9 appeared to be mostly involved in the metabolism and detoxification of xenobiotics such as ß-naphthol, ß-naphthylamine, caffeic acid and gallic acid. SULT3 ST4 showed strong activity toward endogenous compounds such as dehydroepiandrosterone (DHEA), pregnenolone, and 17ß-estradiol. SULT3 ST5 showed weaker, but significant, activities toward endogenous compounds such as DHEA and corticosterone, as well as xenobiotics including mestranol, ß-naphthylamine, ß-naphthol, and butylated hydroxyl anisole (BHA). pH-dependency and kinetic constants of these three enzymes were determined with DHEA, ß-naphthol, and 17ß-estradiol as substrates. Reverse transcription-polymerase chain reaction (RT-PCR) was performed to examine the expression of these three new zebrafish SULTs at different developmental stages during embryogenesis, through larval development, and on to maturity.

A comparative study of the sulfation of bile acids and a bile alcohol by the Zebra danio (Danio rerio) and human cytosolic sulfotransferases (SULTs).

The current study was designed to examine the sulfation of bile acids and bile alcohols by the Zebra danio (Danio rerio) SULTs in comparison with human SULTs. A systematic analysis using the fifteen Zebra danio SULTs revealed that SULT3 ST2 and SULT3 ST3 were the major bile acid/alcohol-sulfating SULTs. Among the eleven human SULTs, only SULT2A1 was found to be capable of sulfating bile acids and bile alcohols. To further investigate the sulfation of bile acids and bile alcohols by the two Zebra danio SULT3 STs and the human SULT2A1, pH-dependence and kinetics of the sulfation of bile acids/alcohols were analyzed. pH-dependence experiments showed that the mechanisms underlying substrate recognition for the sulfation of lithocholic acid (a bile acid) and 5α-petromyzonol (a bile alcohol) differed between the human SULT2A1 and the Zebra danio SULT3 ST2 and ST3. Kinetic analysis indicated that both the two Zebra danio SULT3 STs preferred petromyzonol as substrate compared to bile acids. In contrast, the human SULT2A1 was more catalytically efficient toward lithocholic acid than petromyzonol. Collectively, the results imply that the Zebra danio and human SULTs have evolved to serve for the sulfation of, respectively, bile alcohols and bile acids, matching the cholanoid profile in these two vertebrate species.