Toxicity and detoxification of imidacloprid in mouse ovarian follicles
Neonicotinoids are synthetic nicotine derivates synthesized with high affinity for invertebrate nicotinic acetylcholine receptors (nAChRs) and comparatively low affinity for mammalian nAChRs. They are used in diverse industries ranging from commercial agriculture to veterinary pharmaceuticals, resulting in chronic exposure of nontarget species such as humans, birds, fish, and insect pollinators. Although neonicotinoids are considered insect selective, they can be activated in the environment and within mammals into more toxic metabolites. Imidacloprid (IMI) was the first neonicotinoid synthesized and continues to be the most popular member of the neonicotinoid family today. Mammals are exposed to IMI primarily through ingestion of contaminated food and water as well as through interactions with companion animals. IMI is readily absorbed in the gastrointestinal tract and undergoes phase I and phase II biotransformation in the liver prior to excretion primarily in the urine. In the liver, IMI is subjected to oxidations and reductions by cytochrome P450s (CYPs) and aldehyde oxidases (AOXs). Notably, IMI can be reduced to desnitro-imidacloprid (DNI), a protonated metabolite that binds mammalian nAChRs with much higher affinity than IMI. DNI as well as other redox intermediates are further modified to 6-hydroxynicotinic acid (6HNA) and 6-chloronicotinic acid (6CNA), which are the terminal phase I metabolites. IMI, 6HNA, and 6CNA have been detected in the ovaries of female animals within hours of exposure, verifying that the ovaries are exposed to IMI and IMI metabolites. Although some evidence exists that IMI is a reproductive toxicant, there are many unanswered questions regarding the effects of IMI on the ovaries. Thus, the studies described in my dissertation were designed to test the hypothesis that IMI causes ovarian follicle toxicity via acetylcholine pathways and the ovary itself contributes to this ovotoxicity using its metabolic machinery. In Chapter 3, I compared the effects of IMI and DNI on antral follicle physiology in vitro. Antral follicles were cultured with a vehicle control (DMSO or ultrapure water), IMI (0.2 - 200 µg/mL), or DNI (0.2 - 200 µg/mL) for 48, 72, and 96 hours. Specifically, I evaluated follicle growth, morphology, sex steroid hormone levels, and gene expression of the steroidogenic regulators (Star, Cyp11a1, Cyp17a1, Cyp19a1, Hsd3b1, and Hsd17b1), estrogen receptors (Esr1 and Esr2), and apoptotic factors (Bax and Bcl2). I found that DNI, but not IMI, inhibited follicle growth and caused follicles to rupture in culture compared to control. IMI increased progesterone, whereas DNI decreased estradiol, progesterone, and testosterone compared to control. DNI also increased estradiol levels in the media compared to controls. Both IMI and DNI dysregulated expression of steroidogenic regulators, although the magnitude of change was not enough to result in the observed changes in hormone secretion. In Chapter 4, I tested the hypothesis that ovarian antral follicles metabolize IMI in vitro. Antral follicles were cultured with DMSO or IMI (0.2 - 200 µg/mL) for 48 and 96 hours. At the end of the culture period, media were used for metabolite detection and the follicles were subjected to qPCR to quantify metabolic enzyme gene expression. Environmental controls were also included to differentiate follicular metabolism from environmental breakdown of IMI. Oxidized intermediates, reduced intermediates, and one terminal metabolite were detected in both 48 hour and 96 hour culture media. Oxidized intermediates were not detected in the environmental controls, indicating that antral follicles oxidize IMI in culture. Reduced intermediates were detected in the environmental controls as well as the media cultured with follicles. Interestingly, the media from cultured follicles had more DNI than the environmental controls at 48 hours and less DNI than the environmental controls at 96 hours. At both time points, IMI-urea was detected at higher concentrations than DNI. Furthermore, 6CNA was detectable in the media from cultured follicles at 48 hours and increased 3-fold by 96 hours. IMI- associated CYPs and AOXs have not previously been characterized in the ovaries and more specifically in antral follicles. AOX1, AOX2, AOX3, CYP2D22, and CYP2E1 catalyze IMI reductions and CYP4F18 catalyzes IMI oxidations. IMI induced Cyp2e1 and Cyp4f18 in the follicles cultured for 96 hours compared to the controls. In Chapter 5, I localized nAChR subunit expression in the ovaries and I tested the hypothesis that IMI, and its bioactive metabolite DNI, differentially modulate their expression in immature and mature ovarian follicles. Although some cholinergic signaling pathways have been identified in the ovaries, nAChRs and their subunit compositions have not been characterized within the ovaries [7-9]. I used whole neonatal ovaries and antral follicles to compare expression levels of α and β subunits in immature and mature ovarian follicles. Chrna2, Chrna4, Chrna5, Chrna6, Chrna7, Chrnb1, Chrnb2, Chrnb3, and Chrnb4 were detected in both neonatal ovaries and antral follicles by PCR. Chrna2, Chrna4, and Chrna7 transcripts were localized to granulosa cells and oocytes using RNA in situ hybridization. CHRNA2, CHRNA4, and CHRNA7 were also quantified in ovaries by western blot. To determine whether IMI and DNI affect nAChR subunit expression, I cultured isolated antral follicles and whole neonatal ovaries with a vehicle control (DMSO or ultrapure water), IMI (0.2 - 200 µg/mL), or DNI (0.2 - 200 µg/mL) for 48 hours. The antral follicles and neonatal ovaries were subjected to PCR to quantify α and β subunit expression. Neither IMI nor DNI affected nAChR subunit expression in antral follicles compared to the controls. Both IMI and DNI dysregulated subunit expression in the neonatal ovaries compared to controls. Taken together, the data from my doctoral experiments have contributed to our understanding of the effects of neonicotinoid insecticides on the mammalian ovary.
ovary, follicles, ovarian follicles, antral follicles, pesticide, pesticide toxicology, neonicotinoid, insecticide, imidacloprid, reproductive toxicology, toxicology
University of Illinois - Urbana-Champaign
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