Master of Science in Pharmaceutical Science (MSP), University of Toledo, 2020, Pharmaceutical Sciences (Pharmacology/Toxicology)

Opioid drugs are of great importance in the management of acute and chronic pain conditions. They are recognized as being essential in the management of severe malignant and non-malignant pain (Mercadante, 2015). Pharmacokinetic differences among these drugs contribute to patients having differential responses, including bioavailability, metabolism, and elimination from the body. It is becoming more evident that genetics play a vital role in affecting the metabolism of opioid drugs. Three major enzyme systems, CYP450, UDP-glucuronosyltransferase (UGTs) and SULTs have been shown to be involved in their metabolism. Polymorphisms of these enzyme systems can result in an individual having distinct phenotypes: poor metabolizers which express two nonfunctional alleles, intermediate metabolizers at least one reduced functional allele, extensive metabolizers at least one functional allele, and ultra-rapid metabolizers express multiple copies of the functional allele. These genetic differences are partially explained by single nucleotide polymorphisms, (-SNPs), which encode for molecular entities involved in the pharmacodynamic and pharmacokinetic processes. Understanding how and to what degree the allozymes of different enzyme systems such as the CYPs, UGTs and SULTs affect drug metabolism can add new insights into gene therapy-based approaches and greatly improve the treatment of chronic pain (Peiro, 2016.) My thesis research was focused on the role of human SULTs in the metabolism of opioid drugs. Previous studies have demonstrated that of the eleven known SULTs, SULT1A3 was found to be involved in opioid drug metabolism (Bairam, 2018). By carrying out in vitro assays, we first determined the sulfating activity of SULT1A3 toward opioid drugs and subsequently the effects of SNPs of human SULT1A3/SULT1A4 genes on the enzymatic characteristics of SULT1A3 allozymes in mediating hydromorphone and pentazocine, two commonly used opioid drugs. The results obtained provided va (open full item for complete abstract)

Committee: Ming-Cheh Liu (Committee Chair); Frederick Williams (Committee Member); Caren Steinmiller (Committee Member) Subjects: Pharmaceuticals; Pharmacology

Master of Science in Pharmaceutical Sciences, University of Toledo, 2010, College of Pharmacy

Cytosolic sulfotransferases are phase II drug metabolizing enzymes responsible for carrying out a sulfonation reaction which adds a sulfonyl group to a hydroxyl or amino group of its substrate. These enzymes are believed to be involved with the regulation of endogenous hormones such as neurosteroids as well as xenobiotics. Considering the increasing rates of environmental estrogen exposures, it is important to understand the role that sulfotransferases may play in the early stages of vertebrate development. SULT2 ST1 is a sulfotransferase identified in zebrafish which resembles the SULT2 A1 gene in humans. To establish a link between sulfotransferase expression and developmental toxicity, the SULT2 ST1 gene was knocked down in the zebrafish model. This was accomplished by microinjection of translation blocking morpholino oligonucleotides into <4 cell stage zebrafish embryos. The phenotypic changes observed in the SULT2 ST1 knockdown fish indicate that the absence of the enzyme may be responsible for underdeveloped fin and systemic failure in embryo development phenotypes. Western Blot was used to confirm a reduction in expressed protein in knockdown animals.

Committee: Frederick Williams PhD (Committee Chair); Ming-Cheh Liu PhD (Committee Member); Steven Peseckis PhD (Committee Member) Subjects: Pharmacology; Toxicology

Master of Science in Pharmaceutical Sciences, University of Toledo, 2010, College of Pharmacy

Sulfonation is an important reaction in regulating the biological activities of a variety of endogenous and environmental compounds and is catalyzed by cytosolic sulfotransferases that use PAPS (3'-phosphoadenosine-5'-phosphosulfate) as the sulfonate (SO3-) donor. SULTs are present in mammals and other vertebrates and play an important role in the detoxification of xenobiotics especially environmental estrogens. SULTs are also involved in the biotransformation of endogenous compounds (hormones, steroids) which might be a mechanism for maintaining the homeostasis of these compounds in-vivo. In this study we have attempted to assess the role of the hydroxysteroid sulfotransferase SULT2 ST2 in zebrafish (Danio rerio) development by knocking down the expression of the enzyme using a morpholino. Zebrafish embryos were microinjected with a morpholino which had a sequence complementary to the sequence of the SULT2 ST2 gene. The injections were done when the embryos were in the 1-4 cell stage with three concentrations of the morpholino: 0.5 ng/nL, 1.0 ng/nL and 2.0 ng/nL. The embryos were then observed for survival rates and abnormal phenotypes up to 144 hours post fertilization (hpf). The phenotypes observed were cardiovascular abnormalities such as cardiac edema and irregular heartbeat, abdominal edema, lordosis, notochord deformities, tail deformities and very few cases of craniofacial malformations. At the highest concentration of the morpholino, almost all knockdowns displayed mild to severe cardiac edema which was the specific phenotype at that concentration after the hatching period (48-55 hpf). The extent of knockdown was also determined by western blot experiment and it was found that the knockdown was partial and there was some enzyme present at measurable levels in the embryos. In conclusion, morpholino knockdown of the SULT2 ST2 gene in zebrafish embryos caused several non-specific phenotypes. At the 2.0 ng/nL concentration mild to severe cardiac edema was obser (open full item for complete abstract)

Committee: Frederick Williams PhD (Committee Chair); Ming-Cheh Liu PhD (Committee Member); Steven Peseckis PhD (Committee Member) Subjects: Pharmacology; Toxicology

Master of Science in Pharmaceutical Sciences, University of Toledo, 2010, College of Pharmacy

Sulfation is one of the important Phase II reactions. We identified and characterized two novel zebrafish sulfotransferase SULT1 ST9 and SULT3 ST4. SULT1 ST9 showed strong sulfating activity toward substrates such as acetaminophen and 3 3,3',5-triiodo-L-thyronine. SULT3 ST4 exhibited strong sulfating activity towards substrate DHEA, and weaker activity toward mestranol. pH dependency assays and kinetics assays were used to characterized two enzymes' interactions with these substrates. Acetaminophen and dextromethorphan are widely used in over-the-counter cough and cold medications. Their efficacy and safety for infants and young children remains to be clarified. The present study was designed to use the zebrafish as a model to investigate the potential toxicity of acetaminophen and dextromethorphan during embryonic and larval development. Three sets of zebrafish embryos/larvae were exposed to dextromethorphan at 24 hours post fertilization (hpf), 48 hpf, and 72 hpf, respectively, during embryonic/larval development. Compared with the 48 and 72 hpf exposure sets, the embryos/larvae in the 24 hpf exposure set showed much higher mortality rates which increased in a dose-dependent manner. Morphological effects of dextromethorphan exposure, including yolk sac and cardiac edema, craniofacial malformation, lordosis (curving body trunk), non-inflated swim bladder, and missing gill, were also more frequent and severe among zebrafish embryos/larvae exposed to dextromethorphan at 24 hpf. Moreover, bradycardia (30-70 heart beats/min vs. ~130 heart beats/min for normal control) was observed for the embryos/larvae treated with elevated concentrations of dextromethorphan. Another three sets of zebrafish embryos/larvae were exposed to acetaminophen at 1 hpf, 24 hpf, and 48 hpf, respectively, during embryonic/larval development. Compared with the 24 and 48 hpf sets, the embryos/larvae in the 24 hpf exposure set showed much higher mortality rates which increased in a dose-dependent m (open full item for complete abstract)

Committee: Ming-Cheh Liu (Advisor); Frederick Williams (Committee Member); Zahoor Shah (Committee Member) Subjects: Pharmacology

Master of Science in Pharmaceutical Science (MSP), University of Toledo, 2011, College of Pharmacy

In mammals, sulfation as catalyzed by the cytosolic sulfotransfereases (SULTs) is known to be involved in the metabolism and homeostasis of key endogenous compounds such as thyroid/steroid hormones, as well as in the detoxification of xenobioticsincluding drugs. The present study constitutes part of an overall effort in establishing the zebrafish as a model for studying drug sulfation. By searching GenBank database, we have identified sequences encoding three new zebrafish SULTs. These three novel zebrafish SULTs, designated SULT3 ST4, SULT3 ST5 and SULT1 ST9, were cloned, expressed, purified, and characterized. SULT3 ST4 showed strong activity toward endogenous compound such as dehydroepiandrosterine (DHEA), pregnenolone, and 17β-estradiol. SULT3 ST5 showed weaker, but significant, activities toward endogenous compounds such as DHEA and corticosterone, and xenobiotics including mestranol, β-naphthylamine, β-naphthol, and butylated hydroxyl anisole (BHA). SULT1 ST9, on the other hand, appeared to be mostly involved in the metabolism and detoxification of xenobiotics such as β-naphthol, β-naphthylamine, caffeic acid and gallic acid. pH-dependency and kinetic studies were performed using these three enzymes with DHEA, β-naphthol, β-naphthylamine, and 17β-estradiol as substrates. RT-PCR was carried out to investigate the expression of these three novel zebrafish SULTs during various developmental stages from embryogenesis to maturity.

Committee: Ming-Cheh Liu PhD (Committee Chair); Ezdihar Hassoun PhD (Committee Member); Katherine Wall PhD (Committee Member) Subjects: Pharmacology; Toxicology