High-resolution images show excess DNA double-strand breaks formed in DEHP-exposed nuclei destined to become egg cells. Mid-pachytene nuclei (chromosomes in blue) in germlines dissected from control (DMSO) and DEHP-exposed worms. Sites where DNA double-strand breaks have occurred are marked in magenta (RAD-51 antibody). Scale bar, 3 μm. [This image was created by Luciann Cuenca, Department of Genetics, Harvard Medical School.]

Researchers at Harvard Medical School and the New York State Department of Health have discovered how a common plasticizer that has been linked with human reproductive abnormalities may damage egg production at the molecular level. Studies in Caenorhabditis elegans nematode worms showed that exposure to even low levels of DEHP (di(2-ethylhexyl) phthalate) that might be found in the environment disrupted meiosis—the form of cell division that results in the production of eggs and sperm—and led to changes in chromosome structure, as well as impacting early embryogenesis. The new insights could have implications for those who are pregnant or of reproductive age, for the future regulation of DEHP and other phthalates, and for the ongoing development of “green” chemicals intended to replace phthalates.

“These are completely new findings and hopefully will shed some light as to how this phthalate impacts human reproductive health,” said Monica Colaiácovo, PhD, professor of genetics at the Blavatnik Institute at Harvard Medical School, who is senior author of the team’s paper, which is published in PLOS Genetics, and titled, “Environmentally-relevant exposure to diethylhexyl phthalate (DEHP) alters regulation of double-strand break formation and crossover designation leading to germline dysfunction in Caenorhabditis elegans.”

DEHP is the most abundant plasticizer used in making polyvinyl chloride (PVC)-containing plastics, and about four million tons are produced every year. The chemical is present in a wide range of consumer products, from clothing and personal care products, carpets, and even make-up, pills, and printing inks, the authors explained. While various U.S. federal and state agencies have passed laws limiting the percentage of DEHP and other phthalates in children’s toys, food packaging, drinking water, and other items, the plasticizer can still be found in everyday products ranging from medical devices to rain gear, and shampoo.

DEHP exposure has been associated with reprotoxicity in mammals, including fertility problems in both men and women, the scientists noted. “DEHP exposure is ubiquitous among populations throughout the world and it impairs a myriad of reproductive processes in both females and males.” However, while the effects of DEHP on reproduction have been widely investigated, “… the molecular mechanisms by which exposure to environmentally-relevant levels of DEHP impact on reproductive mechanisms hasn’t been known.” To look into this in more detail, Colaiácovo and colleagues studied the effects of DEHP on female reproductive mechanisms in C. elegans, a common model organism that is used for studying human genetics and biology.

Faithful chromosome segregation during meiosis is key for successful sexual reproduction, the authors explained. In people, an abnormal number of chromosomes (aneuploidy) in sperm and egg cells is responsible for 50% of clinically recognized miscarriages and 4% of stillbirths, as well as infertility and conditions such as Down syndrome. “Mounting evidence from mammalian studies suggests an association between exposures to environmental chemicals and aneuploidy,” the authors noted. However, thousands of man-made chemicals, including endocrine-disrupting chemicals (EDCs) such as phthalates, are highly prevalent in the environment and their impact on meiosis is not fully understood.

C. elegans is a useful system for reproductive studies, the researchers noted. “C. elegans is an ideal system for such studies given that meiosis in this system is well-characterized, this model organism has a rapid life cycle, and its cellular processes and pathways share a high degree of conservation with those found in mammals.” The Colaiácovo lab had previously adapted a strategy in which C. elegans eggs glow green if they develop abnormal numbers of chromosomes. Colaiácovo had previously used these worms to screen dozens of common chemicals to identify those that altered worm egg chromosomes and so were most likely to cause similar abnormalities in humans. DEHP appeared high on the list, along with several other phthalates.

Results from the newly reported study suggest that DEHP impacts on female reproductive mechanisms in multiple ways, disrupting meiosis and so leading to defects during egg formation and very early embryonic development. These effects were evident even when worms were exposed to, and metabolized, low levels of DEHP that were comparable to levels that have been detected in urine samples from the general human population. This indicates that even small amounts of DEHP can disrupt meiosis.

The worm study findings showed that DEHP causes an excessive number of double-stranded breaks (DAB) in DNA as the parental genetic material recombines in the worm eggs. The chemical appears to do this by altering chromosome length and relaxing the normally tightly wound structure of chromatin, exposing more DNA to potential breakage. The phthalate then further compounds the problem by interfering with the system that’s supposed to shut down excess DNA breakage during meiosis.

“Here we show that exposure to environmentally-relevant levels of DEHP induces an excess number of DSBs and we suggest that, at least in part, this is linked to disruption of a negative feedback loop that inhibits DSB formation,” the scientists noted. The overall consequences of DEHP exposure mean that the double-stranded DNA breaks aren’t properly repaired during meiosis, chromosomes have abnormal morphology, eggs contain the wrong number of chromosomes, and embryos are less viable. Problems persisted past meiosis and into the first round of mitotic cell division in the worm embryos. The researchers did not investigate changes beyond this stage of development.

“We were excited to find that [DEHP affects chromosomes] in part by altering chromatin, as suggested by increased length of a structure built on chromosomes during the production of eggs and sperm,” Colaiácovo noted. “Our studies link this alteration to increased levels of DNA double-strand breaks and impaired ability to properly repair these breaks. This underscores the importance of identifying better alternatives for replacing DEHP. “Our future plans involve expanding our analysis to understand the effects of other phthalates and compare this to available (green chemistry) alternatives.”

As the authors concluded, “Taken together, our findings provide new insights into how environmentally-relevant levels of DEHP impair meiosis by linking two non-mutually exclusive processes to increased DSB levels: an impaired negative feedback loop normally set in place to downregulate DSB formation and changes in chromosome structure which may increase chromatin accessibility to DSB-promoting factors.” The reported studies also confirm the usefulness of C. elegans as a model for studying reprotoxicity in this context, the investigators further noted. “Importantly, our study highlights the suitability of C. elegans as a tool to evaluate the effects of phthalate on the germline as it displays conservation of the metabolic pathways mediating phthalate-induced toxicity in humans.”

Interestingly, the results did indicate that the effects of DEHP exposure varied from worm to worm. “Not every worm is affected, nor affected to the same degree,” said Colaiácovo. But that is a feature, not a bug, of the experiment, she suggested. “Not everyone metabolizes DEHP in the same way. The route and duration of exposure, a person’s age and diet, these are just some of the factors that can result in some people being more affected by low-level exposure to a given chemical than other people. You need large numbers to get a complete picture of what a chemical like DEHP may be doing, and we can easily achieve that using worms.”

Worms exposed to the common chemical DEHP developed more problems during egg formation, shown at bottom, than worms not exposed to the chemical, shown at top. [Colaiácovo lab/Harvard Medical School]

More work will be needed to determine whether the worm findings hold true for humans. In the meantime, Colaiácovo will continue to investigate how different chemicals alter reproductive biology. But in the meantime, the team suggested, there is a real need to reduce the use of DEHP. “… coupled to the wide body of research on DEHP reprotoxicity, our results highlight a need for either reducing the use of DEHP as a mainstream plasticizer or identifying an advocating for the use of alternate and safer substitute with the goal of minimizing the risk of human exposure.”

 

 

 

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