Nora J. Deamer

Olfactory toxicity of methimazole: dose-response and structure-activity studies and characterization of flavin-containing monooxygenase activity in the Long-Evans rat olfactory mucosa.

Methimazole is a compound administered to humans for the treatment of hyperthyroidism and is used experimentally as a model substrate for the flavin-containing monooxygenase (FMO) system. Previous results from this laboratory demonstrated that methimazole is an olfactory system toxicant, causing nearly complete destruction of the olfactory epithelium in the male Long-Evans rat following a single ip dose of 300 mg/kg. The present studies were undertaken to determine the dose-response relationship for methimazole-induced olfactory mucosal damage and to determine whether or not similar damage occurs as a result of oral administration, mimicking the relevant route of human exposure. We also investigated the mechanism of olfactory toxicity of methimazole by means of a structure-activity study and began the characterization of the form(s) of FMO present in the olfactory mucosa of the male Long-Evans rat. Dose-response analysis demonstrated that methimazole causes olfactory mucosal damage at doses of 25 mg/kg ip and greater. The results of gavage studies showed that a single oral dose of 50 mg/kg also caused olfactory mucosal damage. Two structurally related compounds, methylimidazole and methylpyrrole, were not olfactory toxicants, suggesting that a reactive intermediate generated in the course of metabolizing methimazole to an S-oxide is the olfactory toxic species. Microsomal incubation studies revealed the presence ofmethimazole S-oxidation activity in olfactory mucosal microsomes at levels comparable to those in liver. An anti-mouse liver FMO antibody reacted on Western blots with olfactory mucosal microsomes. These findings demonstrate a dose-response for the olfactory toxicity of methimazole and suggest that characterization of human olfactory mucosal FMO activity may be necessary to assess the potential for human risk associated with therapeutic exposure to methimazole.

Sonication of bacteria, phytoplankton and zooplankton: Application to treatment of ballast water

We investigated the effect of high power ultrasound, at a frequency of 19 kHz, on the survival of bacteria, phytoplankton and zooplankton, in order to obtain estimates of effective exposure times and energy densities that could be applied to design of ultrasonic treatment systems for ballast water. Efficacy of ultrasonic treatment varied with the size of the test organism. Zooplankton required only 3-9s of exposure time and 6-19 J/mL of ultrasonic energy to realize a 90% reduction in survival. In contrast, decimal reduction times for bacteria and phytoplankton ranged from 1 to 22 min, and decimal reduction energy densities from 31 to 1240 J/mL. Our results suggest that stand-alone ultrasonic treatment systems for ballast water, operating at 19-20 kHz, may be effective for planktonic organisms >100 microm in size, but smaller planktonic organisms such as phytoplankton and bacteria will require treatment by an additional or alternative system.

Vegetative and sexual reproduction in Pfiesteria spp. (Dinophyceae) cultured with algal prey, and inferences for their classification

Abstract Algal-fed clonal zoospore cultures of Pfiesteria piscicida and Pfiesteria shumwayae enabled description of certain conserved morphological and reproductive features. Common modes of reproduction (especially via division cysts) were documented in herbivorous P. piscicida and P. shumwayae using cultures fed algal prey, together with supporting photography and flow cytometric DNA measurements. Other cysts were characterized such as vacuolate cysts in starved P. piscicida cultures and temporary cysts in both species fed algal prey. This study also represents the first report of sexual reproduction in Pfiesteria spp. cultures fed algal prey rather than live fish; the first report of a technique for cell cycle synchronization for these heterotrophic dinoflagellates; and the first information on storage products of cells released from Pfiesteria reproductive cysts. Sexual reproduction in algal-fed P. piscicida clonal cultures was evidenced by fusing gametes, cells with two longitudinal flagella, and nuclear cyclosis. Both isogamous and anisogamous fusions were observed, and resulting cells with two trailing flagella (i.e., planozygotes and planomeiocytes) sometimes comprised ≥50% of the flagellated cells. These cells continued feeding activity and eventually (hours) lost their flagella and formed cysts. Nuclear cyclosis and a subsequent cell division were observed in thin-walled reproductive cysts prior to release of two flagellated cells. One gamete fusion event was also documented in 1 of 20 algal-fed clones of P. shumwayae, with an aplanozygote as the product. We obtained high cell synchrony (≥90% 1C) in the tested cultures using our preferential lysis technique and tracked the decline in lipid content of excysted zoospore populations over time. The data from this study were considered together with previous research to gain insights about relationships between Pfiesteria spp. and other heterotrophic dinoflagellates. Pfiesteria spp. should be regarded as free-living predators rather than parasites because they are prey generalists without demonstrated “host” specificity and their flagellated feeding stages are not morphologically distinct from swimming stages. Although they originally were placed within the Dinamoebales because amoebae can predominate, this study as well as other published research consistently has shown that the dominant stage varies depending on culture conditions, prey type/availability and strains. The peridinoid plate structure of each Pfiesteria species, which thus far has been conserved across culture conditions and strains, supports placement of Pfiesteria spp. within the Peridiniales. At the species level, plate structure (differing by one precingular plate) and molecular data (18S rDNA) indicate that the two Pfiesteria spp. are closely related in comparison to species grouped within other genera.

The Role of Oxidative Enzymes in the Metabolism and Toxicity of Pesticides

Xenobiotics, including pesticides (Hodgson & Levi, 1992, Levi & Hodgson, 1991), are metabolized by many enzymes including: cytochrome P450s (P450); flavin-containing monooxygenases (FMO); prostaglandin synthetase; molybdenum hydroxylases; alcohol dehydrogenase; aldehyde dehydrogenase; esterases; and a variety of transferases, particularly the glutathione transferases. Of these P450 appears to be the most important, followed by the FMO. It should also be remembered that pesticides may serve not only as substrates for these enzymes but, particularly in the case of P450, may serve also as inhibitors and/or inducers. The toxicological implications of these multiple roles are important and are illustrated by our studies of methylenedioxyphenyl (MDP) compounds, of the herbicide synergist tridiphane and the herbicide, dichlobenil, of metabolism in target tissues and portals of entry, and of insect resistance to insecticides.