Chen, Y. et al. (2015) International Worm Meeting "Overlapping and Distinct Effects of Bisphenol A and its Substitute Bisphenol S on Germ Cells."

Allard, P., Yang, X., Chen, Y., Shu, L., Lee, D.

[International Worm Meeting, 2015]

Bisphenol S (BPS), one substitute of Bisphenol A (BPA), is increasingly used in the "BPA-free" plastic manufacturing industry since the toxic effects of BPA have come under heavy scrutiny. Sharing a similar structure and bioactivity with BPA makes BPS a potential threat to human health. However, information about its toxicity is still limited. In this study, the reproductive toxicities of BPA and BPS are investigated and compared using the nematode Caenohabditis elegans (C.elegans). Worms were grown in media with 125, 250 and 500muM BPA and/or BPS. The Bisphenols' effects on adult worms' fertility, germline apoptosis and chromosomal integrity of oocytes were investigated. We observed a dose-response effect for both BPA and BPS with regards to apoptotic germ cell number, embryonic lethality and reduction in brood size, a reflection of fertility. Since meiotic errors could lead to germline apoptosis, aneuploidy formation and even sterility, Bisphenls' effect on the expression of SYP-1 and RAD-51, two critical elements of the meiotic program, were examined. Surprisingly, while both BPA and BPS show ability to impact the formation and disassembly of SYP-1 in the germ cell nucleus, only BPA but not BPS affects the expression and localization of RAD-51 in the germline suggesting a specific effect of BPA on the recombination machinery. To fully reveal the differences between their mechanisms of toxicity, RNA sequencing was also carried out to distinguish the overlapping and distinct effects of BPA and BPS on the transcriptome of germ cells. As a summary, our results show that BPS, like BPA, carries reproductive toxicity. However, some of their mechanisms of toxicity are distinct, especially with regards to the meiotic recombination, and need to be further studied. .

Ferreira, D.W. et al. (2015) International Worm Meeting "Germline defects in mitochondrial cholesterol transporter C. elegans mutants following bisphenol A or low cholesterol exposures."

Allard, P., Ferreira, D.W.

[International Worm Meeting, 2015]

Bisphenol A (BPA) treatment results in abnormal oocyte development in mammalian species as well as in the nematode C. elegans. In C. elegans, these defects are prevented by exposing the worms to cholesterol. We have therefore begun to investigate whether there is an interaction between BPA and normal cholesterol homeostasis in the germline. Previously, we have shown that mammalian homologs to cholesterol transporters (Steroid Acute Regulator Protein; StAR, 18kDa translocator protein; TSPO, and StAR related lipid transfer protein 3; StarD3) mimic germline phenotypes found in BPA-treated worms. Here we show that developing germ cells of these mutants have increases in germ cell nuclei apoptosis and reduced fertility when exposed to no or low cholesterol conditions. Protruding vulva, as well as missing or underdeveloped gonads, are also frequently seen in strl-1 (homologous to StAR) worms. To investigate a possible interaction between BPA and mitochondrial cholesterol transport, wild type and strl-1 worms were treated with BPA. Diakinetic analysis of the -1, -2 and -3 oocytes from control-treated worms reveals an increased incidence of abnormal chromatin arrangement in strl-1 mutant worms compared to wild type (29, 49 and 78 versus 0, 5 and 25 percent). Interestingly, the occurrence among wild type and strl-1 worms is similar following BPA treatment (6, 34 and 71 percent in wild type versus 7, 30 and 73 in strl-1). Additionally, BPA treatment reduced the incidence of protruding vulva (4 vs 24% in controls) and other severe gonad defects (6 vs 31% in controls) in strl-1 mutants. Together, the data suggest that BPA can at least partially rescue the sensitive germline phenotype in strl-1 worms. .

Yonju Ji et al. (2002) East Coast Worm Meeting "Genetic mapping and Characterization of Bisphenol-A Resistant Mutants in Caenorhabditis elegans."

Hyun-Ok Song, Joohong Ahnn, Yonju Ji

[East Coast Worm Meeting, 2002]

Endocrine disrupters are exogenous substances, which can influence endocrine function in humans and other animals. Environmental exposure to these chemicals has been reported to modify sexual development and reproductive function in amphibians, crustacea, and fish. Thus, studies on the effect of endocrine disrupters are important to understand its mechanism. Bisphenol-A(BPA) is one of the endocrine disrupters which can bind to estrogen receptor. First, we studied effect of BPA on the survival of C. elegans. We found that about 90% of the worms died when worms were treated with 90mM BPA plate for 3 hours. In contrast, only 1-2 % of worms died on the control plates. Then we performed EMS mutagenesis to isolate mutants which become resistant to BPA condition. We have isolated mutants showing 70% ~90% survival on BPA treated condition. Currently these candidate mutants are being mapped by using genetic markers. One of these candidate, bpr-1(jh11), has been mapped to LG IV near the dpy-20 locus. This mutant shows a weak Unc phenotype and reproduce normal number of progeny. Another candidate, bpr-2(jh12) shows an egg retention phenotype and shows much decreased brood size. Interestingly, we found that both mutants show moderate resistance to nonylphenol, which is another type of akylphenol. However, these resistance to nonylphenol was not as specific as to BPA, which suggest that these two mutants may have mutations for BPA specific resistance.

Allard, Patrick et al. (2009) International Worm Meeting "C. elegans as a model system to understand environmental influence on germline health."

Allard, Patrick, Colaiacovo, Monica

[International Worm Meeting, 2009]

Although it is clear that environmental toxicants can alter reproductive ability, the dissection of the affected molecular pathways has been particularly challenging. We propose to use C. elegans to investigate the genetic mechanisms of meiotic disruption following environmental exposure. As a proof of principle, we concentrated our research on characterizing the biological effects of Bisphenol-A (BPA), a compound commonly used for the production of polycarbonate-containing plastics. Previous work in mice has shown that exposure to BPA in utero leads to aberrations during prophase of meiosis I including incomplete synapsis, end-to-end chromosome fusions, and an increased number of recombination foci corresponding to elevated recombination frequencies and altered exchange distribution. These defects likely result in increased chromosome nondisjunction as highlighted by the greater number of aneuploid eggs and embryos observed. Assessment of the fertility of adult hermaphrodites in C. elegans following exposure to BPA revealed that BPA causes a dramatic increase in embryonic and larval lethality as well as a decrease in the total number of eggs laid compared to control (exposed to vehicle). A two-fold increase in germ cell apoptosis is detected in BPA-exposed worms suggesting the activation of a DNA damage checkpoint that may stem from a defect in DNA double-strand break repair. Analysis of chromosome morphogenesis in control gonads revealed the presence of 6 DAPI-stained bodies, representing the six pairs of attached homologs in oocytes at diakinesis. In contrast, an aberrant DNA morphology is observed at this stage in BPA-exposed worms. Specifically, bivalents were decondensed and chromatin bridges were apparent. Furthermore, disassembly of the synaptonemal complex (SC) is impaired during late prophase as indicated by an incomplete unloading of SYP-1, a structural component of the SC, from chromosomes in late diakinesis. Taken together, these data demonstrate that exposure to BPA disrupts the meiotic machinery in C. elegans. We are currently further characterizing these meiotic defects by examining recombination levels and the expression of other synapsis components in BPA-exposed animals.

Bowen, J.E. et al. (2019) International Worm Meeting "Effect of bisphenol Z analogs on reproductive function of C. elegans."

Bowen, J.E., Roberts, A., Kuzmanov, A., Zhou, X., Timmons, S.C.

[International Worm Meeting, 2019]

Bisphenol A (BPA), an organic molecule used in different plastic-based products, has been repeatedly linked to neurobehavioral, cardiovascular and reproductive disorders. Because of growing human health concerns, BPA has been increasingly replaced by structurally similar chemicals such as bisphenol S (BPS) and bisphenol Z (BPZ). Whereas numerous studies suggest that BPS may be as toxic as BPA, the impact of BPZ is less understood. To assess its toxicity, we exposed the nematode C. elegans to BPZ at a final concentration of 1 mM throughout their development. Following exposure, we observed marked reduction in the brood size compared to the wild type. In the search for potentially safer alternatives, we synthesized three novel analogs of BPZ. Interestingly, these compounds exhibited even stronger reproductive toxicity, along with impaired larval development. Our finding suggest that BPZ and its analogs are not any safer than BPA and BPS. We are currently investigating potential mechanisms by which these reproductive defects arise.

Allard, Patrick (2019) International Worm Meeting "Dissecting the epigenetic mechanisms of environmental memory and transgenerational reproductive dysfunction in C. elegans."

Allard, Patrick

[International Worm Meeting, 2019]

How artificial environmental cues are biologically integrated and transgenerationally inherited is still poorly understood. Here, we investigate the mechanisms of inheritance of reproductive outcomes elicited by the several common environmental exposures including to ethanol and the highly prevalent plastic-manufacturing chemical Bisphenol A in C. elegans. We show that both exposures cause a dose-dependent derepression of an epigenomically silenced transgene in the germline although the effect for ethanol is less pronounced than that of BPA. We also show that at the F3 generation, both chemicals lead to an increase in reproductive dysfunctions including an increase in germline apoptosis and embryonic lethality. For BPA, several lines of evidence obtained through ChIP-seq, histone modification quantitation, and immunofluorescence assays revealed that this effect is associated with a reduction of the repressive marks H3K9me3 and H3K27me3 in whole worms and in germline nuclei in the F3. Furthermore, targeting of the Jumonji demethylases JMJD-2 and JMJD-3/UTX-1 restores H3K9me3 and H3K27me3 levels, respectively, and it fully alleviates the BPA-induced transgenerational effects. Finally, investigation of the reproductive phenotype indicate a pervasive transgenerational disruption of the recombination machinary including an alteration of ZHP-3 and COSA-1 foci number in late pachytene. Together, our results demonstrate the central role of repressive histone modifications in the inheritance of reproductive defects elicited by common environmental chemical exposures.

Barrere-Cain, Rio et al. (2019) International Worm Meeting "Epigenetic Sensitivity to Chemical Exposure in the Aging C. elegans Germline."

Truong, Lisa, Allard, Patrick, Camacho, Jessica, Barrere-Cain, Rio, Chen, Yichang

[International Worm Meeting, 2019]

How an epigenome's sensitivity to chemical exposure changes with age is not fully understood. This is significant for the germline where the genome is kept relatively silent through repressive histone marks such as H3K9me3 (histone 3 lysine 9 trimethylation) and H3K27me3 (histone 3 lysine 27 trimethylation). Understanding how epigenetic sensitivity changes with age will inform how age should be accounted for in chemical risk assessments. To investigate the relationship between age and epigenetic sensitivity we used immunofluorescence to quantify changes in H3K9me3 and H3K27me3 levels with age in C. elegans germlines. We performed immunofluorescence for H3K27me3 and H3K9me3 at days 1, 3, 5, and 7 of adulthood, with 5-7 replicates and fluorescence quantified in 3-4 gonads per replicate. Variation in H3K27me3 levels increases with age, suggesting a decrease in epigenetic homeostatic control with germline age. Next, we asked whether maternal age affects transmission of a chemical exposure to future generations. We used a green fluorescent protein (GFP) reporter C. elegans strain, NL2507, where GFP expression indicates levels of chromatin accessibility in C. elegans germlines. Under healthy conditions the GFP reporter is silenced in the germline however, with exposure to bisphenol A (BPA), transcriptional regulation is lost and GFP is expressed (Camacho et al., 2018). We exposed C elegans to 500uM BPA during different 48hr windows in their adult lifespan and analyzed GFP levels in F1 and F3 progeny (30 worms per condition). F3 BPA descendants had GFP expression that was double that of their respective control group, indicating that BPA affects the epigenetics of descendants not directly exposed. Furthermore, the F3 from older mothers had higher levels of GFP expression than the F3 from younger mothers. This suggests that older germlines may be more sensitive to chemical exposures because they are more likely to transmit chemically induced epigenetic perturbations to future generations. The epigenetic sensitivity of older germlines is relevant to humans as we tend to have children later in life. Furthermore, understanding how age impacts epigenetic sensitivity will allow us to more accurately assess the safety of chemicals for both exposed individuals and their descendants.

Camacho, Jessica et al. (2015) International Worm Meeting "Examining environmental effects on the germline epigenome."

Hosohama, Linzi, Camacho, Jessica, Allard, Patrick, Lundby, Zachary, Clark, Amander

[International Worm Meeting, 2015]

The high number of compounds to be tested, the diversity of toxicity endpoints, concentrations, and combinations of chemicals, all provide a challenge in our ability to assess the safety of chemicals. Of particular importance is the potential effect of chemicals on the germline epigenome, which can alter biological processes over several generations. While the cases of DES, vinclozolin and BPA provide evidence of epigenetic effects, there is a great need to explore the influence of environmental compounds on the epigenome and, especially, to develop methods that allow us to quickly and efficiently examine this question. We have established the use of the genetic model system, the worm Caenorhabditis elegans, as a relevant model for epigenetic and reproductive toxicity assessment. By taking advantage of the C. elegans genetic tools, we propose to comprehensively identify chemicals for their ability to disrupt the germline chromatin. To this aim, we are making use of a worm strain where GFP is specifically epigenetically silenced in the germline. We previously showed that valproic acid, a well-known mammalian histone deacetylase inhibitor, disrupts the germline epigenetic state leading to the de-silencing of the transgene in the germline. Our current experiments test this concept further by exposing the worms to environmental compounds. We tested vinclozolin and BPA to analyze a disruption in maintenance and/or establishment of epigenetic marks. Young adult worms were exposed to the compounds for 48 hours, and three subsequent generations were followed and analyzed. Results indicate that de-silencing effects last for at least three generations (up to three-fold compared to our DMSO control). Furthermore, most worms showing de-silencing in the first, second and third generation originate from worms showing de-silencing in the parental exposed generation indicating that the effect is inherited. Thus, we conclude that valproic acid, vinclozolin, and BPA disrupt the germline epigenome over several generations in a heritable fashion. Additionally, in concomitant assays, we are working with the lab of Dr. Amander Clark, using mice primordial germ cell-like cells (PGCLCs) in a mammalian validation approach. Together, we hope to establish a novel in vitro germline differentiation assay to screen for chemicals that affect germline quality.

Truong, L. et al. (2019) International Worm Meeting "Examining the transgenerational effects of environmental cues in C. elegans."

Camacho, J., Vo, D., Truong, L., Allard, P.

[International Worm Meeting, 2019]

In sexually reproducing organisms, germ cell development is vital for faithful genome and epigenome transmission across generations. Recent studies have shown that germ cell development is affected by different environmental toxicants, resulting in a decrease in germ cell health and number. Here, we examine and compare the transgenerational impact and mechanisms of two prevalent toxicants, the plastic chemical Bisphenol A and ethanol. Both have well-described impacts on the developing fetus; however, their effects on developing germ cells and subsequent generations are less explored. We analyze the transgenerational effects of both compounds in Caenorhabditis elegans. We hypothesize that exposure disrupts the epigenetic machinery in germ cells, causing changes in histone modifications, fertility defects, and germline dysfunction in a transgenerational manner. First, we showed that BPA exposure causes a transgenerational germline chromatin desilencing coupled with a reduction and redistribution of histone H3K9me3 and H3K27me3. We showed that the alteration of repressive histone mark levels is required for the observed transgenerational increase in germline apoptosis and embryonic lethality. Similar to BPA, ethanol exposure at human-relevant doses also causes transgenerational chromatin desilencing and germline dysfunction, although to a lower extent than BPA's. Current work examines whether the same histone demethylases involved in BPA's transgenerational responses also apply to ethanol. This project identified BPA's and ethanol's transgenerational effect on the germline epigenetic machinery and reproductive health. We hope to further understand how it can induce germline dysfunction, carrying important implications for human reproductive health in the context of environmental exposures.

Moore, C. et al. (2019) International Worm Meeting "Evaluating microcystin-LR induced transgenerational toxicity."

Alllard, P., Moore, C., Blumenkrantz, M.

[International Worm Meeting, 2019]

Persistent drought conditions and increased nutrient water pollution run off perpetuate the public health risk of microcystin-producing harmful algal blooms. Well-established acute hepatotoxins, microcystins (MCs) are known serine/threonine protein phosphatase (PP) inhibitors and can alter cell signaling, resulting in a wide variety of clinical signs, including death. Of the over 100 MC congeners, MC-LR is the most commonly detected and studied. Recent studies highlight the reproductive system as a target organ. Multigenerational studies in animals looking at the offspring of exposed parents have demonstrated changes in the F1 generation, including altered neurodevelopment, growth, and oxidative stress markers. In the alternative model Caenorhabditis elegans (C. elegans) MCs induce germline apoptosis and decrease brood size at P0, making it an ideal model to study the epigenetic impact of MCs several generations. L4s are exposed to a range of environmentally relevant concentrations MC-LR (0.1-100 g/L) for 48 hours with food in liquid. The C. elegans strains N2 (wildtype) and NL2507, carrying an integrated low-complexity, highly repetitive array composed of a transgene coding for a fusion product between nuclear-localized LET-858 and GFP (pkIs1582[let-858::GFP; rol-6(su1006)]), are used. For NL2507, the transgene is expressed in somatic cells but it is epigenetically silenced in the germline via accumulation of the repressive marks H3K9me3 and H3K27me3. Prior exposure studies using bisphenol A (BPA) have established this method to evaluate transgenerational toxicity. Exposed P0 worms are recovered and each generation is evaluated for multi- (F1) and trans- (F3) generational toxicity without further MC exposure. Chromosome remodeling and epigenetic histone modifications in the germline are evaluated using immunofluorescence, germline stress is monitored through desilencing via loss of repressive histone regulation, and germline and somatic cell toxicity is evaluated through apoptotic, growth, and behavioral endpoints. This study helps establish the transgenerational reproductive and somatic cell toxicity of MCs and the role of phosphorylation in the epigenetic repression of the germline.