Synthesis and reproductive toxicity of bisphenol A analogs with cyclic side chains in Caenorhabditis elegans

Abstract

Accumulating evidence has shown that bisphenol A (BPA) affects not only the growth and development of reproductive tissues but also disrupts meiosis. Meiotic disturbances lead to the formation of aneuploid gametes, resulting in the inability to conceive, pregnancy loss, and developmental disabilities in offspring. In recent years, increasing health concerns led manufacturers to seek BPA alternatives. In response, BPA analogs have been prepared and investigated in a variety of toxicity-related studies. Despite hopes that these analogs would prove less harmful than BPA, published data show that these alternatives continue to pose a significant risk to human health. In this study, we synthesized two less investigated BPA analogs with cyclic side chains, bisphenol Y (BPY) and bisphenol Z (BPZ), and evaluated their reprotoxic potential using Caenorhabditis elegans. C. elegans were cultured on nematode growth medium plates containing a 1 mM concentration of the dimethyl sulfoxide-dissolved bisphenols. The uptake of the chemicals was via two major routes: ingestion and cuticle diffusion. Following exposure, we evaluated fertilized egg count, germline apoptosis, and embryonic lethality—three parameters previously shown to reliably predict the reprotoxic potential of bisphenols in mammals. Our results indicated that both BPY and BPZ had a significant impact on fertility, resulting in increased germline apoptosis and a reduced number of progeny, without affecting the embryonic viability. After comparison with commercially relevant BPA and bisphenol S, our findings imply that BPA analogs with cyclic side chains, BPY and BPZ, adversely affect meiotic fidelity, resulting in diminished reproductive capacity.

Keywords

bisphenol A bisphenol A analogs reprotoxicity meiosis

Introduction

Bisphenol A (BPA) is an age-old chemical that was synthesized as early as 1891 (Vogel, 2009). Its commercial properties were first realized in the 1950s when chemists synthesized the first epoxy resins using BPA (Bilyeu et al., 2005). The strong adhesive properties of epoxy resins quickly resulted in their widespread use as protective coatings in a variety of industrial applications. In the 1970s, the use of BPA was extended to the production of polycarbonates, plastics strong enough to replace steel and clear enough to replace glass. These new BPA-containing polymers found many uses as components of electronics, automobiles, food packaging, safety equipment and more. By the late 1970s, the market for BPA-containing epoxy resins and polycarbonate plastics was booming, with BPA production reaching half a billion pounds annually in the United States (Greiner et al., 2004). In recent years, approximately 5–6 billion pounds of BPA was produced worldwide on an annual basis (CDC National Biomonitoring Program, 2017).

The widespread global use of BPA led to its ubiquitous presence in the environment and continued BPA exposure in the general population. BPA has been detected in urine, blood, and various tissues in the majority of the human population, including children (Liao and Kannan, 2013; Vandenberg et al., 2007). Over the past two decades, BPA has been linked to a number of health-related disorders, including infertility (Almeida et al., 2018; Rochester, 2013). Accumulating evidence has shown that BPA affects not only the growth and development of reproductive tissues, but also meiotic fidelity by preventing normal chromosome alignment and segregation (Campen et al., 2018; Can et al., 2005; Eichenlaub-Ritter et al., 2008; Hunt et al., 2003; Lenie et al., 2008; Machtinger and Orvieto, 2014; Susiarjo et al., 2007; Wang et al., 2016). BPA has been shown to exert its genotoxic activity during meiosis by interfering with the repair of deliberately induced DNA double-strand breaks (DSBs) (Allard and Colaiácovo, 2010; Brieño-Enríquez et al., 2012; Lee et al., 2013; Prokešová et al., 2020). The repair of these lesions allows the exchange of genetic material and ensures accurate chromosome segregation during meiosis, which is critical for maintaining genome integrity and creating gametes (eggs and sperm) with the correct number of chromosomes. If left unrepaired, DSBs may result in the loss of genetic material and recombination failure. Recombination defects prevent the proper alignment and segregation of chromosomes in the first meiotic division (Cannan and Pederson, 2016; Gray and Cohen, 2016; Murakami and Keeney, 2008). Mounting evidence suggests that the genotoxic effects of BPA may contribute to increased infertility, recurrent miscarriages, and the incidence of inherited developmental disabilities in the general population (Machtinger and Orvieto, 2014; Rochester, 2013).

In recent decades, numerous reports of health concerns and implications of BPA exposure due to its ubiquitous industrial usage have prompted the search for safer alternatives. Manufacturers have increasingly been replacing BPA in plastic packaging with structurally similar analogs that share the same basic chemical structure of two phenolic rings connected at the para position by a central atom, most commonly carbon or sulfur (Figure 1) (Morgan and Clifton, 2021; Mustieles et al., 2020; Pelch et al.,2019). Among the array of different bisphenol derivatives, bisphenol S (BPS) has been the most frequently used commercial alternative (Morgan and Clifton, 2021; Owczarek et al., 2018). Despite hopes that BPS would prove less harmful than BPA, recent studies indicate that BPS also poses a significant risk to human health and the environment (Moon and Kyong, 2019; Rochester and Bolden, 2015). Similar to BPA, BPS has been shown to exert significant reprotoxicity by adversely affecting meiotic progression and causing chromosomal abnormalities in reproductive cells (Chen et al., 2016; Cosentino et al., 2022). Our understanding of the reprotoxic potential of other structurally related BPA alternatives is far from complete. While recent studies suggested that the BPA analogs interfered with endocrine signaling and disrupt hormonal balance, not much is known about their effect on meiosis (Pelch et al., 2019).

Studies utilizing the nematode Caenorhabditis elegans have been shown to reliably predict the reprotoxic potential of bisphenols in mammals (Allard and Colaiácovo, 2010; Allard et al., 2013). Consistent with findings in mammalian models, studies in C. elegans have shown impaired meiotic progression resulting in unrepaired DSBs and subsequent apoptosis of the defective germ cells in animals exposed to BPA and BPS (Allard and Colaiácovo, 2010; Chen et al., 2016). Unlike mammalian assays, C. elegans-based assays are rapid and inexpensive, providing an easy and reliable method for preliminary comparative reprotoxic screening of bisphenols (Allard et al., 2013). In this study, we synthesized two BPA analogs with cyclic side chains, bisphenol Y (BPY) and bisphenol Z (BPZ), and used C. elegans to compare their toxicities to the reprotoxic bisphenols, BPA and BPS (Figure 2).

Materials and methods

Synthesis of bisphenol Y and bisphenol Z

General Methods. BPA, BPS, and all chemicals used in the synthesis of additional bisphenol analogs were purchased from Sigma-Aldrich and used without further purification. Analytical thin-layer chromatography (TLC) was performed on polyester-backed TLC plates pre-coated with silica gel containing a fluorescent indicator (200 μm layer thickness, 5–17 μm particle size). Developed TLC plates were visualized using a shortwave UV lamp (254 nm). Reaction mixtures were purified using a Biotage® Isolera™ Spektra One automated flash chromatography system. Nuclear magnetic resonance (NMR) data was recorded using an Agilent MR-400 MHz spectrometer in the Lumigen Instrument Center at Wayne State University. Chemical shifts are reported in parts per million (ppm) and were referenced to the dimethyl sulfoxide (DMSO)-d₆ solvent peak at 2.50 ppm. High-resolution mass spectra (HRMS) were recorded using a Thermo Scientific LTQ Orbitrap XL™ mass spectrometer in the Lumigen Instrument Center at Wayne State University.

Synthetic Methodology. An adapted electrophilic aromatic substitution protocol was used to prepare BPY and BPZ (Gregor, 2012). Phenol (1.127 g, 12 mmol), a cyclic ketone (3 mmol), and concentrated aqueous HCl (0.5 mL) were combined in a round-bottomed flask with a magnetic stir bar. The resulting reaction mixture was fitted with a water-cooled reflux condenser and heated in a silicone oil bath at 70°C while stirring for 48 h. Reactions were monitored via TLC using a 9:1 dichloromethane/ethyl acetate eluent, which revealed the formation of a new bisphenol product at a lower R_f value than phenol when visualized using 254 nm UV light. After 48 h, the reaction mixture was placed in a −20°C freezer until it was purified via automated flash chromatography using a C18 reversed-phase column and HPLC-grade water and acetonitrile as the mobile phase.

Characterization of BPY. ¹H NMR (DMSO-d₆, 400 MHz): δ (ppm) 9.07 (s, 2H, OH), 6.91 (d, 4H, Ph), 6.62 (d, 4H, Ph), 2.15 (m, 4H, CH₂), 1.53 (m, 8H, CH₂); ¹³C{¹H} NMR (DMSO-d₆, 100 MHz): δ (ppm) 154.52, 141.18, 127.66, 114.53, 47.92, 29.19, 23.55; HRMS (ESI) m/z: [M-H]^- calcd for C₁₉H₂₁O₂, 281.1547; found, 282.1547.

Characterization of BPZ. ¹H NMR (DMSO-d₆, 400 MHz): δ (ppm) 9.08 (s, 2H, OH), 7.03 (d, 4H, Ph), 6.63 (d, 4H, Ph), 2.13 (br s, 4H, CH₂), 1.42 (br s, 6H, CH₂); ¹³C{¹H} NMR (DMSO-d₆, 100 MHz): δ (ppm) 154.57, 139.00, 127.51, 114.76, 44.10, 36.55, 25.86, 22.51; HRMS (ESI) m/z: [M-H]^- calcd for C₁₈H₁₉O₂, 267.1391; found, 267.1391.

C. elegans maintenance

C. elegans strains were cultured on nematode growth medium (NGM) supplemented with Escherichia coli OP50 as a food source according to standard protocols (Stiernagle, 2006). The animals were maintained at 20°C. The following strains were used in the study: N2 Bristol strain (wild type) and MD701 [bcIs39 (lim-7p::ced-1::GFP + lin-15(+))].

Bisphenol treatment

The bisphenol analogs used in the study were dissolved in DMSO and mixed with molten NGM before plate pouring to facilitate efficient absorption (Zheng et al., 2013). The treatment plates did not contain cholesterol because this chemical was shown to mask the damage caused by BPA and BPS exposure in C. elegans (Allard and Colaiácovo, 2010; Chen et al., 2019). C. elegans uptake chemicals via two routes: ingestion and cuticle diffusion (Hartman et al., 2021). The final plate concentration of bisphenols was 1 mM, whereas the final plate concentration of the vehicle, DMSO, was 0.1%. Previous studies in C. elegans have shown that reprotoxic effects using the parameters chosen in this study can be very subtle when the animals are exposed to bisphenol concentrations lower than 1 mM (Allard and Colaiácovo, 2010; Chen et al., 2016). Therefore, a 1 mM bisphenol concentration was selected to provide sufficiently robust reproductive effects, facilitating a reliable comparative assessment of bisphenol analogs. In all the experiments, 0.1% aqueous DMSO served as a control. All experiments were performed at 20°C and were independently conducted at least three times, with 3–5 animals per trial. The final numbers differed between treatment groups because in some of the bisphenol treatments worms more often crawled away onto the plastic and desiccated.

Longevity assay

The wild-type worms were continuously exposed to a 1 mM concentration of bisphenol analogs starting at the embryonic stage (Figure 3(a)). Worms were scored for survival by their response to mechanical stimuli and live animals were transferred to new bisphenol-containing plates every other day. Lifespan was defined as the length of time from the embryonic stage until the animals were scored as dead.

Fertility assessment

The wild-type worms were exposed to a 1 mM concentration of bisphenol analogs from the embryonic stage until early adulthood (Figure 3(b)). Once worms reached the L4 stage, they were singled on new bisphenol treatment plates and exposed to the bisphenol analogs for additional 48 h. To facilitate easy monitoring, once the bisphenol treatment was done, the singled worms were transferred to new NGM plates without cholesterol every day. The animals were followed throughout their reproductive period (approximately 5 days from the beginning of the egg-laying period) until they reached reproductive senescence (marked by the appearance of unfertilized eggs). During the egg-laying period, laid fertilized eggs and successfully hatched larvae were counted using a dissecting light microscope.

Germline apoptosis assay

Germline apoptosis was assessed by counting the number of defective germ cells culled by apoptosis in a C. elegans strain containing GFP-tagged CED-1. The CED-1 fluorescent marker labeled the surface of engulfed apoptotic cells, allowing detection using fluorescence microscopy (Lu et al., 2009). L4-stage larvae were synchronized by picking and treated with 1 mM concentrations of bisphenol analogs for 24 h (Figure 3(c)). Following exposure, the number of nuclei undergoing apoptosis were counted using a Zeiss Axiophot fluorescence microscope with AxioVision 4.5 software.

Statistical analysis

Longevity assay: The survival function of each worm population was estimated using the Kaplan–Meier method and statistical analysis was performed using a log-rank test.

Fertility assessment and germline apoptosis assay: All statistical analyses were performed using the one-way ANOVA with Tukey’s post hoc test. The mean differences with a p value less than 0.05 were considered statistically significant.

Results

Synthesis, yields, and characteristics of bisphenol Y and bisphenol Z

Bisphenol Z was prepared by adapting an electrophilic aromatic substitution reaction protocol involving phenol, cyclohexanone, and a concentrated HCl catalyst (Figure 4) (Gregor, 2012). Reaction stoichiometry, temperature, and time were varied to improve the yield. A longer reaction time of 48 h produced a significantly higher yield of 97%. Using the same reaction parameters, the synthesis of BPY produced a lower yield of 13% despite multiple attempts to improve this reaction, which is presumably the result of the conformation and/or steric hindrance of the cycloheptanone reactant. Both BPY and BPZ were purified using an automated flash chromatography system fitted with a C18 reversed-phase column and characterized via HRMS and ¹H and ¹³C{¹H} NMR spectroscopy to ensure compound identity and high purity. Results from the synthesis of BPY and BPZ are summarized in Table 1.

Table 1.

Yields and characteristics of synthesized BPA analogs.

Cyclic Ketone	Product name	Product characteristics	Yield (%)
Cyclohexanone	Bisphenol Z	White powder	97
Cycloheptanone	Bisphenol Y	Light brown powder	13

Effect of bisphenol Y and bisphenol Z exposure on longevity

To evaluate the overall toxicity of the BPY and BPZ analogs, we first assessed their impact on life expectancy. All bisphenol-treated animals reached adulthood at the same time as animals on control plates, demonstrating no overt toxicity on animal development. As shown in Figure 5, animals on the DMSO control plates lived 13.7 ± 0.43 days on average. BPA and BPY exposure significantly reduced the animals’ lifespan compared to the control (BPA: 11.8 ± 0.29 days; BPY: 10.9 ± 0.50 days; p < 0.05). Whereas the average lifespan of animals exposed to BPZ was extended (14.9 ± 0.38 days; p < 0.05), BPS did not have any significant impact on the longevity of the exposed worms (13.4 ± 0.53 days).

Fertility assessment following exposure to bisphenol Y and bisphenol Z

To evaluate the effect of the bisphenol analogs on fertility, we first quantified the number of eggs laid following treatment. As shown in Figure 6(a), the DMSO-treated worms produced 205.6 ± 10.3 eggs on average. The worms exposed to the bisphenol analogs produced fewer embryos: 113.2 ± 14.9 in BPA-exposed worms; 159.6 ± 22.1 in BPS-exposed worms; 133.5 ± 16.3 in BPY-treated worms, and 167.2 ± 16.5 in BPZ-treated worms. A statistically significant reduction in the number of eggs laid was observed in BPA- and BPY-exposed animals (p < 0.05).

To investigate the effect of BPY and BPZ on embryonic viability, we determined the percentage of unhatched laid eggs. In all treatment groups, most of the laid fertilized eggs hatched (Figure 6(b)). The percentages of unhatched eggs were as follows: 0.8 ± 0.2% in DMSO-treated worms, 2.7 ± 0.9% in BPA, 1.7 ± 0.9% in BPS, 3.5% ± 1.4 in BPY, and 1.8% ± 1.3 in BPZ.

To investigate whether reduced fertility was due to abnormal meiosis progression, we assessed the number of gametes undergoing apoptosis. Similar to BPA and BPS, BPY and BPZ exposure caused a significant increase in the number of apoptotic nuclei in the germline (Figure 7). We observed a 1.8-fold increase in the number of apoptotic nuclei in BPA-exposed worms, a 1.9-fold increase in BPS-exposed worms, a 1.6-fold increase following BPY exposure, and a 1.5-fold increase following BPZ exposure relative to the DMSO control.

Discussion

In this study, we compared the reproductive toxicity of BPA, BPS, BPY, and BPZ using the model organism C. elegans. Exposure to each of the four bisphenol analogs resulted in decreased fertility, suggesting that BPY and BPZ analogs impact the reproductive ability of C. elegans in a similar fashion as the well-known reprotoxins BPA and BPS.

Previous studies in C. elegans have shown that some bisphenol analogs could promote aging and affect the life expectancy of exposed animals (Hyun et al., 2021; Tan et al., 2015). Unlike BPS, BPA exposure has been shown to shorten the C. elegans lifespan (Hyun et al., 2021; Tan et al., 2015). Although BPA adversely affects the animal longevity, the length of their reproductive period is not significantly affected and they live past reproductive senescence (Hyun et al., 2021; Tan et al., 2015). In the present study, the BPY analog decreased the life expectancy of the exposed animals. However, their reproductive lifespan was not affected, allowing us to investigate the effect of BPY on the reproductive capacity of C. elegans.

In this study, all of the evaluated bisphenol derivatives, BPA, BPS, BPY and BPZ, caused a significant increase in germline apoptosis. In addition, we observed a trend towards a decrease in the overall fertilized egg count in all four bisphenols tested, with BPA and BPY reaching significance compared to the control. Our findings are consistent with previous research in C. elegans reporting that BPA and BPS exposures result in an increased incidence of germline apoptosis, a reduced number of mature eggs, and a reduced brood size (Allard and Colaiácovo, 2010; Chen et al., 2016). Both BPA and BPS have been shown to interfere with the worm’s ability to repair meiotic DSBs, leading to an increased incidence of recombination defects and germ cell death mediated by CEP-1/p53, a protein essential for triggering DNA damage-response checkpoint (Allard and Colaiácovo, 2010; Chen et al., 2016). The adverse effects of BPA and BPS have been also studied in mammalian animal models. Previous studies in mice, cows and pigs, for example, showed that BPA and BPS exposure disrupt oogenesis by causing meiotic arrest at different stages due to synapsis and recombination abnormalities (Susiarjo et al., 2007), an increase in DSBs (Lee et al., 2013; Prokešová et al., 2020), meiotic spindle abnormalities (Campen et al., 2018; Can et al., 2005; Eichenlaub-Ritter et al., 2008; Wang et al., 2016; Žalmanová et al., 2017), and chromosome misalignment (Campen et al., 2018; Eichenlaub-Ritter et al., 2008; Hunt et al., 2003; Wang et al., 2016; Žalmanová et al., 2017). Although a fraction of the affected oocytes is able to resume meiosis following arrest, most of the oocytes never complete the maturation process and die by apoptosis, decreasing the number of mature oocytes (Lenie et al., 2008; Wang et al., 2016).

Since the meiotic disturbances lead to chromosome missegregation and aneuploidy, we also assessed embryonic lethality. While we observed a slight increase in embryonic lethality in worms exposed to BPA and BPY, the observed differences were not statistically significant. Although previous studies in C. elegans reported an increase in embryonic lethality following exposure to BPA and BPS (Allard and Colaiácovo, 2010; Chen et al., 2016, 2019; Tan et al., 2015), the observed frequencies of embryonic lethality varied across different publications. In some cases, differences in the length of bisphenol exposure and the type of vehicle used in the study (DMSO vs ethanol) may explain the variability in embryonic lethality reported among different research groups. However, in some cases, the same conditions resulted in different embryonic survival frequencies. For instance, two studies observing embryonic lethality following a 4 days-exposure to 500 μM BPA dissolved in 100% ethanol reported substantially different results: 15% embryonic lethality (Chen et al., 2016) vs. more than 90% embryonic lethality (Allard and Colaiácovo, 2010). In addition, work by our group and others shows significant variability in embryonic lethality in animals even within the same sample. Studies in mammalian model organisms also show inconsistent results when it comes to the occurrence of aneuploidy. BPA-exposed oocytes have been shown to have multiple meiotic defects that could lead to chromosomal aberrations in gametes and embryos (Campen et al., 2018; Can et al., 2005; Eichenlaub-Ritter et al., 2008; Hunt et al., 2003; Susiarjo et al., 2007; Wang et al., 2016; Žalmanová et al., 2017). However, meiotic disturbances do not always increase the incidence of aneuploidy, which might be due to an early check point mechanism triggered by higher BPA concentrations (Eichenlaub-Ritter et al., 2008). The oocytes exposed to lower BPA doses appear to be able to avoid the checkpoint more often, contributing to the formation of aneuploid gametes and, consequently, an increased incidence of chromosomal aberrations in embryos (Lenie et al., 2008). Therefore, a plausible explanation for the variability in the embryonic lethality seen across different C. elegans studies might be an uneven uptake of the chemical from the medium due to the cuticle barrier.

Taken together, our findings demonstrate that BPA, BPS, BPY, and BPZ affect the reproductive function of C. elegans in a similar fashion, implying that prolonged exposure to BPY and BPZ, bisphenol analogs with cyclic side chains, may lead to reproductive health risks and are thus not promising substitutes for BPA and BPS. Future studies will focus on BPA analogs with structurally diverse acyclic side chains and phenolic ring substitutions.

Footnotes

Acknowledgments

The authors thank the Lumigen Instrument Center staff at Wayne State University for mass spectrometry and NMR spectroscopy service and support.

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by the C. elegans strains were provided by the Caenorhabditis Genetics Center (CGC), which is funded by NIH Office of Research Infrastructure Programs (P40 OD010440). The synthesis of bisphenol analogs was supported by HHMI Inclusive Excellence grant #52008705 (S.C.T., program director).

ORCID iDs

Isabella Burke

Aleksandra Kuzmanov

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