Assessing how benzyl butyl phthalate exposure impairs the germline using Caenorhabditis elegans

Abstract

Epidemiological and experimental data suggest that exposures to Endocrine Disrupting Chemicals (EDCs) are linked to reproductive health issues including infertility, miscarriages, stillbirths, and birth defects. However, few studies have focused on the direct effects of EDCs on the germline. My dissertation addresses this important issue using the nematode C. elegans, a genetically tractable model organism that has a well characterized meiotic program, shares a high degree of gene conservation with humans, and is highly predictive of human reprotoxicity. I specifically assess the effects of benzyl butyl phthalate (BBP), a widely used plasticizer found in various consumer, medical, and building products, and one of six phthalates identified in human maternal urine samples proposed to act as an EDC. In chapter 2, using a high-throughput screening method and a combination of cytological and genetic tools, my studies revealed that BBP elicits a non-monotonic dose response in C. elegans. Mass spectrometry analysis of whole worm lysates determined that, as in humans, BBP is rapidly metabolized into monobutyl phthalate (MBP) and monobenzyl phthalate (MBzP) in worms. Moreover, this analysis showed that exposure to a 10 μM dose results in internal levels that are comparable to concentrations detected in human biological samples. Subsequent analyses showed that exposure to 10 μM BBP: (1) leads to elevated mitotic and meiotic DNA double-strand break (DSB) formation throughout the germline; (2) causes activation of a p53/CEP-1-dependent DNA damage checkpoint in late pachytene; (3) alters meiotic progression, resulting in an increase in the number of nuclei in a leptotene/zygotene-like state persisting into pachytene; and (4) increases chromosome morphology defects in oocytes at diakinesis. RNA sequencing and oxidative stress analyses revealed that BBP exposure results in elevated oxidative stress in the germline, potentially underpinning the significant increase in DNA damage, meiotic dysfunction and loss of genomic integrity. Another important and understudied issue is whether exposure to EDCs might sensitize our bodies to additional genotoxins. Given the increase in DSBs observed throughout the germline following BBP exposure, I also explored whether BBP exposure had the potential to sensitize the germline to other forms of DNA lesions. In chapter 3, I conducted a series of DNA damage sensitivity assays using known genotoxins. These agents included nitrogen mustard, to assess the effects of DNA inter-strand crosslinks, camptothecin, to assess the effects of single-strand breaks, UVC, to assess the effects of pyrimidine dimers, hydroxyurea, to inhibit DNA synthesis and induce replication fork stalling/arrest, and gamma-irradiation as another measure of assessing the effects of DSBs. My data shows that BBP has the potential to sensitize the germline to specific types of DNA damage arising from DSBs and pyrimidine dimers. Taken together, this dissertation identifies the effects of BBP on the female germline, discusses the potential basis for these effects, and highlights the efficacy of using C. elegans to rapidly assess and predict the effects of exposures to environmental toxicants on higher order organisms.