ALTERED GENE EXPRESSION: A MECHANISM OF REPRODUCTIVE TOXICITY IN ZEBRAFISH (DANIO RERIO) EXPOSED TO BENZO[a]PYRENE

ALTERED GENE EXPRESSION: A MECHANISM OF REPRODUCTIVE TOXICITY IN ZEBRAFISH (DANIO RERIO) EXPOSED TO BENZO[a]PYRENE by: Jennifer L. Hoffmann Although reductions in 17ß-estradiol and vitellogenin concentrations have been consistently observed in fish exposed to polycyclic aromatic hydrocarbons (PAHs), an understanding of the mechanism behind these changes has yet to be fully elucidated. The overall objective of the present research was to gain a better understanding of the effect of PAHs on transcription of genes involved in reproduction. The first objective of this research was to characterize the expression of follicle stimulating hormone receptor (FSH-R) and luteinizing hormone receptor (LH-R) and four key steroidogenic enzymes (P450-side chain cleavage (P450scc), P450 17α-Hydroxylase/17-20 lyase (P450c17), 20β-Hydroxysteroid Dehydrogenase (20β-HSD), P450-aromatase (P450arom)) in oocytes and whole ovaries at different stages of development in zebrafish to gain a better understanding of basic transcriptional regulation of ovarian development and maturation. All genes examined were differentially expressed in pre-, early, mid, and late-vitellogenic oocytes. In contrast, only LH-R was differentially expressed at 0, 48, and 96 hours after spawning. The results of the first experiment provided evidence that transcription of gonadotropin receptors and steroidogenic enzymes may be involved in regulating ovarian development and maturation. Additional objectives of this project were 1) to determine if exposure to waterborne benzo[a]pyrene (B[a]P) (0, 1.5, or 3.0 μg/L) for 61 days affected reproduction in zebrafish and 2) to determine if waterborne exposure to B[a]P affected transcription of gonadotropins, steroidogenic enzymes, activin, vitellogenin, or estrogen receptor β. A 50% and 48% reduction in total egg output was observed in the 1.5 μg/L and 3.0 μg/L treatments as compared to controls, respectively. Although not significant, there was a trend towards a decrease in the number of females that spawned following exposure to 3 μg B[a]P/L. Cytochrome P4501A1 (CYP1A1) mRNA was significantly increased in heads of B[a]P-exposed zebrafish whereas it was not elevated in livers of B[a]P-exposed fish. An increase in P450aromB, vitellogenin, and 20β-HSD mRNA occurred in fish exposed to 3.0 μg B[a]P/L as compared to the controls. The results from this study demonstrate that reproduction in zebrafish is affected by waterborne exposure to B[a]P and that transcription of P450aromB, vitellogenin, and 20β-HSD may be one mechanism of B[a]P-induced reproductive toxicity. ALTERED GENE EXPRESSION: A MECHANISM OF REPRODUCTIVE TOXICITY IN ZEBRAFISH (DANIO RERIO) EXPOSED TO BENZO[a]PYRENE A DISSERTATION Submitted to the Faculty of Miami University in partial fulfillment of the requirements for the degree of Doctor of Philosophy Department of Zoology by Jennifer L. Hoffmann Miami University Oxford, Ohio 2004 Dissertation Director: James T. Oris TABLE OF CONTENTS Page LIST OF FIGURES . iv LIST OF TABLES . vii CHAPTER 1: GENERAL INTRODUCTION . 1 CHAPTER 2: Methods for mRNA Analysis: RNA Isolation, Reverse Transcription, and Optimization of Conventional and Real-time PCR Methods 2.