A high-throughput approach to identify reproductive toxicants among environmental chemicals using an in vivo evaluation of gametogenesis in budding yeast Saccharomyces cerevisiae

Abstract

Background: Environmental chemical exposures are likely making important contributions to current levels of infertility and its increasing incidence. Yet the US produces high volumes of industrial chemicals for which there is limited data on their potential human reproduction toxicity. Current assays typically used in policy and regulatory settings involve costly and time- consuming whole-animal rodent tests which limit the rapidity with which one can assess the thousands of chemicals yet to be tested. Objective: Our aim was to develop a fast and reliable strategy to evaluate a large number of chemicals for reproductive toxicity by developing a high-throughput toxicity assessment using the yeast S. cerevisiae. Methods: Yeast are chronically exposed to each environmental chemical at two doses, 30 μM and 100 μM, in a 96-well plate-based format throughout gametogenesis. Non-gametes are removed and chemicals are washed away before gamete viability is measured using absorbance at 600 nm to produce growth curves. The difference in time at half-maximal saturation with and without exposure is used to determine the extent of reproductive toxicity. Results: We validated our assay using bisphenol A (BPA), a well-established mammalian reproductive toxicant. We find that BPA in yeast has similar detrimental effects in meiosis as shown in worms and mammals. Competition assays with BPA analogs reveal that two of out of 19 BPA analogs examined (bisphenol E and 17b-estradiol) show synergistic effects with BPA at doses tested and none show antagonistic effects. Out of 179 additional environmental chemicals, we designated 57 chemicals as reproductively toxic. Finally, by comparing chemicals in our cohort that have been evaluated for reproductive toxicity in mammalian studies, we find a statistically significant association between toxic chemicals in yeast and mammals. Conclusion: We show that a high-throughput assay using yeast may be a useful approach for rapidly and reliably identifying chemicals that pose a reproductive risk.

Screening and characterization of 133 physiologically-relevant environmental chemicals for reproductive toxicity

“…First, 111 chemicals were tested at two concentrations (30 μM and 100 μM) based on the good predictive value of the screens at the highest concentration [ 21 ], with four biological repeats each performed in experimental duplicates. Of these 111 chemicals, a subset of 61 chemicals were further tested at all four concentrations (10 μM, 30 μM, 50 μM and 100 μM) with five biological repeats performed in duplicate in order to facilitate future comparison with a comparable yeast assay [ 22 ] ( Table S1 ). Due to solubility limitations, 21 chemicals were tested at lower concentrations than those indicated ( Table S1 ).…”

Analyzing high-throughput assay data to advance the rapid screening of environmental chemicals for human reproductive toxicity

“…Some of the chemicals were further assessed for the specific stage of meiosis affected and identification of genes and key meiotic processes involved. The yeast assay is based on detecting gamete viability using absorbance measurements to assess the growth of viable gametes through growth curves that shift to the right when the pool of gametes is reduced post exposure [23]. After non-gametes are removed in the assay, the number of viable gametes can be determined by measuring the mitotic growth of living cells that remain in the culture via optical densities at 600 nm (OD 600 ) on microplate readers.…”

“…Previous research has shown that these two assays can predict reproductive toxicity rapidly and cost-effectively. Yeast models are an established platform for screening effects on gametogenesis [26], as demonstrated by research from our collective group, which showed how this model can be used to screen for reproductive toxicants, including BPA and its replacement analogs [23]. Similarly, the C. elegans assay has been shown to perform well as a rapid screen for germline toxicants through the induction of aneuploidy [25], a relevant predictor of decreased litter size and ovarian defects in mammalian studies [24].…”

“…To that end, we assessed the performance of two non-mammalian in vivo HTP assays that are directly relevant to germline function (i.e., aberrant chromosome segregation or aneuploidy) and fertility: 1) yeast-based ( Saccharomyces cerevisiae) in vivo assay of meiosis (and mitosis) [23]; and 2) nematode ( Caenorhabditis elegans ) in vivo assay which captures chemically-induced meiotic and chromosome errors [24,25]. Previous research has shown that these two assays can predict reproductive toxicity rapidly and cost-effectively.…”

Screening and characterization of 133 physiologically-relevant environmental chemicals for reproductive toxicity

“…; https://doi.org/10.1101/2024.03. 22.584808 doi: bioRxiv preprint takes advantage of the naturally hermaphroditic (XX) state of C. elegans and the naturally low occurrence of males (<0.2%). The appearance of males in the population is often a result of meiotic segregation errors, i.e.…”