Exposure to benzyl butyl phthalate (BBP) leads to increased double-strand break formation and germline dysfunction in Caenorhabditis elegans

Endocrine disrupting chemicals (EDCs) have been linked to various health problems including effects on reproductive health. Benzyl butyl phthalate (BBP) is a commonly used plasticizer found in many consumer products that has been shown to act as an EDC and affect human reproduction and development. However, how it might impact the germline and affect the specialized cell division program of meiosis, which results in the formation of haploid gametes (i.e. eggs and sperm), remained unclear. Here, using the nematode C. elegans, we show that BBP exposure at levels within the range of what is detected in humans, impairs accurate chromosome segregation which can result in aneuploidy. Similar to mammals, BBP exposure in C. elegans results in a non-monotonic dose response, whereby lower doses result in the strongest effects, and BBP is efficiently metabolized, underscoring the use of this model system for gaining mechanistic insights into how this EDC impacts the germline. BBP results in increased levels of DNA double-strand breaks (DSBs), activation of a DNA damage checkpoint, and altered chromosome morphology in exposed germlines. We propose that BBP exposure alters gene expression and results in increased oxidative stress, resulting in increased DSBs, thereby compromising chromosome morphology and accurate segregation. Overall design: Following 24-hour exposure to either 0.1% DMSO alone or BBP dissolved in DMSO, 1,200 worms per condition were washed five times with M9, then collected in 500 µl of Trizol (Invitrogen) for RNA extraction, and frozen at -80°C. There were four biological replicates per treatment. RNA purification was performed using the Direct-zol RNA Miniprep Plus kit (Zymo Research) following manufacturer's instructions. RNA concentrations, as well as the 260/280 and 260/230 ratios, were assessed using a Nanodrop (Thermo Fisher Scientific). The RNA was prepared for sequencing with the Kapa mRNA HyperPrep kit (Roche) and sequenced on a NovaSeq 6000 using an SP 2x50bp run (Illumina) by the Bauer Core Facility at Harvard University.