Mammalian Monooxygenases and Environmental Chemicals

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Title: Mammalian Monooxygenases and Environmental Chemicals
Author: Karoly, Edward Douglas
Advisors: Dr. Ernest Hodgson, Committee Co-Chair
Dr. Randy L. Rose, Committee Co-Chair
Abstract: A cDNA clone of mouse FMO2 was obtained as an expressed sequence tag (EST) isolated from a female mouse kidney cDNA library from the I.M.A.G.E. consortium (I.M.A.G.E. CloneID 1432164). Complete sequencing of the EST derived a nucleotide sequence for mouse FMO2 that contains 112 bases of 5' flanking region, 1608 bases of coding region and 309 bases of 3' flanking region. This FMO2 sequence encoded a protein of 536 amino acids and shows 87 and 86% homology to rabbit and human FMO2, respectively. The mouse FMO2 was found to be heat stable and demonstrated optimal activity at a relatively high pH of 10.5. The expressed FMO2 showed catalytic activity towards methimazole, thiourea, trimethylamine and the insecticide phorate. The S-oxidative metabolism of phorate to phorate sulfoxide and methiocarb to methiocarb sulfoxide was investigated in human liver microsomes. Use of specific inhibitors for CYPs and FMOs demonstrated that metabolite production was primarily due to CYP activity. Subsequent screening of 16 cDNA-expressed human CYP isoforms and 3 cDNA-expressed human FMO isoforms and their metabolites were analyzed by HPLC. Of the isoforms examined, CYP 1A2, 3A4, 2B6, 2C9*1, 2C18, 2C19, 2D6*1 and FMO1 had the highest rates of phorate sulfoxide production. For methiocarb, CYP 1A1, 1A2, 3A4, 2B6, 2C9*1, 2C19, 2D6*1 and FMO1 were the isoforms with the highest rates of methiocarb sulfoxide production. Additionally, a cDNA clone of mouse FMO4 was also obtained as an EST isolated from a male mouse kidney cDNA library from the I.M.A.G.E. consortium (I.M.A.G.E. CloneID 4234388). Complete sequencing of the EST derived a nucleotide sequence for mouse FMO4 that contains 402 bases of 5' flanking region, 1683 bases of coding region and 31 bases of 3' flanking region. This FMO4 sequence encodes a protein of 561 amino acids and shows 80% homology to both the rabbit and human FMO4. However, the expression of mouse FMO4 was unsuccessful which was identical to previous attempts from other laboratories to express rabbit and human FMO4.
Date: 2003-08-19
Degree: PhD
Discipline: Toxicology
URI: http://www.lib.ncsu.edu/resolver/1840.16/5580


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