Despite their utility, on a regular basis necessities—cosmetics, meals packaging, plastics—can include endocrine-disrupting chemical substances (EDCs). These stealthy invaders can slip into the physique by way of pores and skin absorption, inhalation, and ingestion, quietly stirring up well being points.
Take benzyl butyl phthalate (BBP), for instance.1 This additive makes plastic merchandise versatile and sturdy, however publicity to BBP can disrupt hormonal steadiness and mess with human reproductive well being.2,3 Whereas animal research trace at its results on egg cell growth and early embryos, BBP’s affect on the earliest phases of meiosis stays largely understudied.4
Monica Colaiácovo research germline upkeep and chromosome inheritance throughout meiosis.
Monica Colaiácovo
This motivated Monica Colaiácovo, a molecular and cell biologist at Harvard Medical Faculty, to discover BBP’s results on the early phases of replica in Caenorhabditis elegans. Her staff’s findings, revealed in PLOS Genetics, show that publicity to BBP ranges which are similar to these detected in people triggered DNA strand breaks in C. elegans, leading to egg cells with the flawed variety of chromosomes.5 These insights provide a extra detailed understanding of the early occasions of BBP publicity and its penalties in animal replica.
Most C. elegans are hermaphrodites able to self-fertilization, sometimes producing hermaphroditic offspring with two X chromosomes. Males, with their single X chromosome, come up as a result of errors throughout X chromosome segregation and make up simply 0.1 to 0.2 p.c of the inhabitants. Colaiácovo sought to grasp how EDCs affect this course of, and the proportion of males within the worm inhabitants.
Many EDCs exhibit non-monotonic dose responses, the place the connection between dose and impact is just not linear—the next dose doesn’t at all times produce a stronger impact. To discover this, the researchers used a high-throughput technique to display screen numerous doses of BBP—1, 10, 100, and 500μM—to find out which dose triggered errors in X-chromosome nondisjunction, or separation, after 24 hours. They used worms with a mutation in a cuticle collagen gene, which makes it simpler for EDC compounds to permeate the worm, in an effort to analyze decrease publicity doses.
For his or her experiments, the researchers chosen worms within the late larval stage, proper earlier than reaching maturity which corresponds to peak reproductive exercise. To trace male embryos, the researchers tagged worms with a inexperienced fluorescent protein (GFP) managed by a male-specific promotor after which used stream cytometry to type the embryos. They discovered that 10μM was the bottom dose of BBP that produced the best X-chromosome nondisjunction results, which elevated male progeny in C. elegans.
“Should you’re interfering with meiosis, the frequency with which these X chromosomes is not going to partition correctly goes up considerably,” defined Colaiácovo. “In most of the meiotic mutants, as a substitute of 0.2 p.c males, you find yourself with 30 to 40 p.c males. So, it’s extremely straightforward to see,” she stated.
Utilizing mass spectrometry, the researchers discovered that C. elegans metabolized BBP in the same method as people, breaking BBP down into two main metabolites. Based mostly on the dose response experiment, the staff examined 10 and 100μM for physiologically related ranges of BBP throughout the worms. Solely the 10μM dose resulted in BBP and metabolite concentrations similar to ranges beforehand reported within the urine of pregnant ladies, umbilical twine samples, and amniotic fluid.6
To zoom in on adjustments in chromosome group throughout meiosis, the researchers used nuclear membrane and DNA injury protein stains to trace nuclear habits. Since these occasions sometimes observe a well timed schedule, any delays can point out defects on this course of. “What we noticed on this case was that you simply had these nuclei that persevered. We name them ‘laggers’ as a result of they’re lagging behind,” stated Colaiácovo. “Then, very late into meiotic prophase, they’re nonetheless carrying the sign [in the nuclei], which ought to already be gone.” This stalled meiotic development coincided with the activation of a DNA injury checkpoint, triggered by double-strand break formation, resulting in defects like chromosome fraying and fragmentation. Consequently, this led to elevated embryonic lethality.
In comparison with management worms (left), the egg cells of BBP-treated C. elegans exhibited chromosome fragments.
Ayana Henderson
With BBP publicity rising germ cell dying and impaired chromosome integrity, she sought to determine the genes related to this course of. Utilizing RNA sequencing, they recognized 344 genes that had been differentially expressed in worms uncovered to 10μM BBP. Amongst these downregulated had been genes essential for extracellular matrix processes and egg cell integrity.
“What was very placing for us once we had been taking a look at these genes is that they prompt that there may be activation of an oxidative stress response,” stated Colaiácovo. To dig deeper, the staff used a C. elegans pressure carrying a fluorescent tag linked to an oxidative stress reporter. After they analyzed germline cells, the staff noticed a rise in fluorescence, an indication that BBP publicity ramped up oxidative stress. This increase in stress led to DNA injury, compromised genomic integrity, and disrupted the precision of meiotic chromosome segregation.
Patrick Allard, a developmental biologist on the College of California, Los Angeles, who was not concerned on this research, remarked that BBP is thought to trigger oxidative stress that’s related to reproductive toxicity, however, “This paper goes additional by taking a look at particular phases of germ cell differentiation and offering detailed details about the method of meiotic recombination and the way impacted it’s by BBP publicity.”
Colaiácovo plans to discover the results of BBP within the male germline, and whether or not there’s a sexual dimorphism influencing reproductive well being. “There’s this entire different thrilling new route, and that was one thing that you would be able to’t essentially predict, and so we had been very excited to see this and to attempt to perceive why that is taking place,” she stated.
“This research offers you some perception on the mechanism, and it serves as extra data so individuals could make extra educated selections based mostly on this work, to attempt to see what may be in a specific product that they are utilizing, and maybe select an alternate,” remarked Colaiácovo.
