Molecular Genetics and Enzyme Regulation of Epoxide Hydrolases in the Cabbage Looper, Trichoplusia ni.

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Title: Molecular Genetics and Enzyme Regulation of Epoxide Hydrolases in the Cabbage Looper, Trichoplusia ni.
Author: Anspaugh, Douglas Day
Advisors: Randy L. Rose, Committee Member
Michael D. Tomalski, Committee Member
Deborah M. Thompson, Committee Member
Ernest Hodgson, Committee Member
R. Michael Roe, Committee Chair
Abstract: A full-length epoxide hydrolase (EH) cDNA (TmEH-2) was isolated from a Trichoplusia ni cDNA library made from the digestive system of fifth (last) stadium larvae. When TmEH-2 was compared to a previously reported T. ni EH cDNA (TmEH- 1) from fat body, the two were 67 and 73% identical at the nucleic acid and amino acid levels, respectively. In a phylogenetic tree of EHs inferred from amino acid sequence alignments, TmEH-1, TmEH-2 and other known insect EHs were more closely related to the microsomal than soluble EHs of other organisms. The tree not only separated microsomal from soluble EHs, but also grouped all insect EHs into one microsomal cluster. Contrary to earlier conceptions, it was determined in multiple sequence alignments that not all membrane-bound microsomal EHs (mEHs) contain identical amino acid residues at the sites of the proposed catalytic triad. While the T. ni EHs possessed the typical Asp-Glu-His triad of mEHs, the residues were Asp-Asp-His at the same positions in two of three previously reported Drosophila melanogaster mEH cDNAs. This soluble-like EH triad also was discovered in mEHs from two bacterial species. To determine the tissue specificity of TmEH-1 and TmEH-2 expression, total RNA from T. ni fat body or gut was assayed by Northern blot using TmEH-1 and TmEH-2 gene-specific probes. Although TmEH-1 and TmEH-2 were isolated from fat body and gut, respectively, each was expressed in both tissues. Also, both EHs were expressed in a similar, age-specific manner. TmEH-1 was subcloned into a baculovirus system for in vivo expression in T. ni larvae. Following injections with non-occluded virus, the time to lethality of the TmEH-1 baculovirus was shorter than the non-transformed, control virus. At 72 h post-injection, the cumulative mortality for the TmEH-1 and control baculoviruses was 95 and 41%, respectively. The mechanism of accelerated toxicity of the TmEH-1 baculovirus currently is unknown. Juvenile hormone (JH) III esterase and JH III EH in vitro activity was compared in whole body T. ni homogenates at each stage of development. While activity of both enzymes was detected at all ages tested, JH esterase was significantly higher than EH activity at all time points except day three of the fifth stadium. For both enzymes, activity was highest in eggs. Interestingly, adult virgin females had 4.6- and 4.0-fold higher JH esterase and EH activities, respectively, than adult virgin males. JH III metabolic activity also was measured in whole body homogenates of fifth stadium T. ni that were fed a nutritive diet (control) or starved on a non-nutritive diet of alphacel, agar and water. With larvae that were starved for 6, 28 and 52 h, EH activity per insect equivalent was 48, 5 and 1%, respectively, of the control insects. At the same time points, JH esterase levels in starved T. ni were 29, 4 and 3% of insects fed nutritive diet. Selected insect hormones and xenobiotics were administered topically or orally to fifth stadium larvae for up to 52 h, and the effects on whole body EH and JH esterase activity were then analyzed. JH III induced JH III esterase as high as 2.2-fold, but not JH III EH activity. The JH analog, methoprene, increased both JH esterase and EH activity as high as 2.5-fold. The JH esterase inhibitor, 3-octylthio-1,1,1-trifluoropropan-2-one (OTFP), had no impact on EH activity. The epoxides, trans- and cis-stilbene oxide (TSO and CSO) each induced EH between 1.9- and 2.0-fold. TSO did not alter JH esterase levels when topically applied, but oral adminstration reduced activity to 70% of the control at 28 h, and then induced activity 1.8-fold at 52 h after treatments began. CSO did not have an impact on JH esterase activity. Phenobarbital increased EH activity by 1.9-fold, but did not change JH esterase levels. Clofibrate and cholesterol 5α,6α-epoxide had no effect on EH. JH esterase activity also was not affected by clofibrate, but cholesterol 5α,6α-epoxide was inhibitory to JH esterase, lowering activity to 60-80% of the control. The biological significance of these results is discussed.
Date: 2003-07-31
Degree: PhD
Discipline: Toxicology

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