Despite their utility, everyday essentials—cosmetics, food packaging, plastics—can contain endocrine-disrupting chemicals (EDCs). These stealthy invaders can slip into the body through skin absorption, inhalation, and ingestion, quietly stirring up health issues.
Take benzyl butyl phthalate (BBP), for example.1 This additive makes plastic products flexible and durable, but exposure to BBP can disrupt hormonal balance and mess with human reproductive health.2,3 While animal studies hint at its effects on egg cell development and early embryos, BBP’s impact on the earliest stages of meiosis remains largely understudied.4
This motivated Monica Colaiácovo, a molecular and cell biologist at Harvard Medical School, to explore BBP’s effects on the early stages of reproduction in Caenorhabditis elegans. Her team’s findings, published in PLOS Genetics, demonstrate that exposure to BBP levels that are comparable to those detected in humans caused DNA strand breaks in C. elegans, resulting in egg cells with the wrong number of chromosomes.5 These insights offer a more detailed understanding of the early events of BBP exposure and its consequences in animal reproduction.
Most C. elegans are hermaphrodites capable of self-fertilization, typically producing hermaphroditic offspring with two X chromosomes. Males, with their single X chromosome, arise due to errors during X chromosome segregation and make up just 0.1 to 0.2 percent of the population. Colaiácovo sought to understand how EDCs influence this process, and the proportion of males in the worm population.
Many EDCs exhibit non-monotonic dose responses, where the relationship between dose and effect is not linear—a higher dose does not always produce a stronger effect. To explore this, the researchers used a high-throughput method to screen various doses of BBP—1, 10, 100, and 500μM—to determine which dose caused errors in X-chromosome nondisjunction, or separation, after 24 hours. They used worms with a mutation in a cuticle collagen gene, which makes it easier for EDC compounds to permeate the worm, in order to analyze lower exposure doses.
For their experiments, the researchers selected worms in the late larval stage, right before reaching adulthood which corresponds to peak reproductive activity. To track male embryos, the researchers tagged worms with a green fluorescent protein (GFP) controlled by a male-specific promotor and then used flow cytometry to sort the embryos. They found that 10μM was the lowest dose of BBP that produced the greatest X-chromosome nondisjunction effects, which increased male progeny in C. elegans.
“If you're interfering with meiosis, the frequency with which these X chromosomes will not partition properly goes up significantly,” explained Colaiácovo. “In many of the meiotic mutants, instead of 0.2 percent males, you end up with 30 to 40 percent males. So, it's very easy to see,” she said.
Using mass spectrometry, the researchers found that C. elegans metabolized BBP in a similar manner as humans, breaking BBP down into two primary metabolites. Based on the dose response experiment, the team examined 10 and 100μM for physiologically relevant levels of BBP within the worms. Only the 10μM dose resulted in BBP and metabolite concentrations comparable to levels previously reported in the urine of pregnant women, umbilical cord samples, and amniotic fluid.6
To zoom in on changes in chromosome organization during meiosis, the researchers used nuclear membrane and DNA damage protein stains to track nuclear behavior. Since these events typically follow a timely schedule, any delays can indicate defects in this process. “What we saw in this case was that you had these nuclei that persisted. We call them ‘laggers’ because they're lagging behind,” said Colaiácovo. “Then, very late into meiotic prophase, they're still carrying the signal [in the nuclei], which should already be gone.” This stalled meiotic progression coincided with the activation of a DNA damage checkpoint, triggered by double-strand break formation, leading to defects like chromosome fraying and fragmentation. Consequently, this led to increased embryonic lethality.
With BBP exposure increasing germ cell death and impaired chromosome integrity, she sought to identify the genes associated with this process. Using RNA sequencing, they identified 344 genes that were differentially expressed in worms exposed to 10μM BBP. Among those downregulated were genes crucial for extracellular matrix processes and egg cell integrity.
“What was very striking for us when we were looking at these genes is that they suggested that there might be activation of an oxidative stress response,” said Colaiácovo. To dig deeper, the team used a C. elegans strain carrying a fluorescent tag linked to an oxidative stress reporter. When they analyzed germline cells, the team observed an increase in fluorescence, a sign that BBP exposure ramped up oxidative stress. This boost in stress led to DNA damage, compromised genomic integrity, and disrupted the precision of meiotic chromosome segregation.
Patrick Allard, a developmental biologist at the University of California, Los Angeles, who was not involved in this study, remarked that BBP is known to cause oxidative stress that is associated with reproductive toxicity, but, “This paper goes further by looking at specific stages of germ cell differentiation and providing detailed information about the process of meiotic recombination and how impacted it is by BBP exposure.”
Colaiácovo plans to explore the effects of BBP in the male germline, and whether there is a sexual dimorphism influencing reproductive health. “There's this whole other exciting new direction, and that was something that you can't necessarily predict, and so we were very excited to see this and to try to understand why this is happening,” she said.
“This study gives you some insight on the mechanism, and it serves as additional information so people can make more educated decisions based on this work, to try to see what might be in a particular product that they're using, and perhaps choose an alternative,” remarked Colaiácovo.
- Cao XL. Phthalate esters in foods: Sources, occurrence, and analytical methods. Compr Rev Food Sci Food Saf. 2010;9(1):21-43.
- Jurewicz J, et al. Human urinary phthalate metabolites level and main semen parameters, sperm chromatin structure, sperm aneuploidy and reproductive hormones. Reprod Toxicol. 2013;42:232-241.
- Henderson AL, Colaiácovo MP. Exposure to phthalates: Germline dysfunction and aneuploidy. Prenat Diagn. 2021;41(5):610-619.
- Jiang Y, et al. Nicotinamide mononucleotide restores oxidative stress-related apoptosis of oocyte exposed to benzyl butyl phthalate in mice. Cell Prolif. 2023;56(8):e13419.
- Henderson AL, et al. Exposure to benzyl butyl phthalate (BBP) leads to increased double-strand break formation and germline dysfunction in Caenorhabditis elegans. PLoS Genet. 2024;20(10):e1011434.
- Huang Y, et al. Phthalate levels in cord blood are associated with preterm delivery and fetal growth parameters in Chinese women. PLoS One. 2014;9(2):e87430.
