Soil and groundwater contain a variety of chemical mixtures originating from the manufacturing industry of modern materials. Xenobiotics are defined as non-endogenous substances that can be found in an organism, for example, pesticides and environmental pollutions. The constant exposure to these xenobiotics poses a health risk to humans and animals, e.g., as carcinogens or endocrine disruptors. However, we lack knowledge about how many of these chemicals affect the body, and more research is needed to determine how these xenobiotics interfere with physiological mechanisms. One category of xenobiotics are plasticizers, such as bisphenol A (BPA). Bisphenol is a monomer discovered in the 1890’s used in the plastic manufacturing industry since the 1950’s. Due to the modern lifestyle and its demand for cheap and sustainable materials, the production of plastic is higher than ever, with an estimated world production of 335 million tons in 2015 (1). Polycarbonate and epoxy resins are the main demand for BPA. Polycarbonate is a robust and durable type of plastic with many applications, ranging from building materials to medical devices and epoxy resins are mainly used as coatings inside food and beverage containers, dental sealants and thermal receipt paper.
The first health concerns about BPA rose in the late 1990’s when BPA was found to be an endocrine disruptor (2) and could advance puberty and stimulating prolactin release in mice (3,4). BPA has a similar structure to the synthetic estrogen receptor agonist diethylstilbestrol (3) and interacts with the nuclear estrogen receptor α and β with low affinity (5,6). Also, BPA binds to the transmembrane estrogen receptor GPR30 (7) as well as the estrogen-related receptor γ (ERRγ) (8), and is equipotent with the endogenous estradiol in its ability to initiate rapid non-genomic responses from membrane receptors (9). In addition to its estrogen-like activity, BPA also binds to the androgen receptor (10), and the thyroid receptor (11) where it acts as an antagonist. Today, a variety of harmful effects of BPA have been reported, including reproductive disturbance, metabolic disruption, and carcinogenic effects. Indeed, BPA is associated with decreased fertility in both men and women. Women undergoing in vitro fertilization showed a poorer ovarian response, reduced fertilization and implantation failure that correlated with higher serum levels of BPA.
Moreover, BPA levels in urine significantly correlated with lower estradiol levels (12–14). In men, BPA is associated with reduced sperm quality (15) and male workers in BPA manufacturing factories self-reported significantly lower sexual function, i.e., erectile function and sexual desire, than controls (16,17). Furthermore, BPA has been detected in amniotic fluid and fetal plasma, indicating passage over the placenta (18,19), interfering with neural signaling and brain development (20). The developing brain is sensitive to non-endogenous estrogen (i.e., xenoestrogens) exposure, including BPA. Animal data shows disturbed neural development in rats, including underdevelopment of the forebrain (21). Moreover, children whose mothers were exposed to BPA during pregnancy showed increased aggression, anxiety, and depression (22,23). The interaction between BPA and the estrogen receptor is suspected to have a role in cell proliferation, apoptosis, and migration of cells(24). BPA might thereby contribute to cancer development and progression in hormone-related cancers, for example, breast, prostate, and ovarian cancers and endometrial carcinoma. Fetal exposure to BPA in rodent models showed increased tumorigenesis (25). However, the underlying mechanisms are poorly understood. Due to these discoveries, several countries applied restrictions in the early 21st century limiting the BPA levels of food packaging and prohibited its use in infant feeding bottles (EU regulation 321/2011). From the 21st century, a group of alternative chemicals was adopted in the industry, for example, bisphenol S (BPS), Bisphenol F (BPF), and bisphenol A diglycidyl ether (BADGE).
BADGE is an epoxy resin monomer obtained by O-Alkylation of bisphenol and epichlorohydrin. In the body, BADGE is rapidly metabolized into BADGE-H2O, BADGE-2H2O, BADGE-4OH, and BADGE-2Cl (26,27). Interestingly, BPA is one of the metabolites of BADGE (28) which indicates a toxicological association between BADGE and BPA. Due to the widespread use of epoxy resins, BADGE can be found almost everywhere, and the general population is exposed through the indoor air (29), canned food (30), and drinking water (31). BADGE and its hydrolytic derivates can be detected in human urine, plasma and adipose fat (32,33). BADGE has a similar estrogenic activity as BPA. However, in vitro binding assay shows no binding to the human ER α receptor, suggesting that non-nuclear receptors are involved in the estrogenic response (26). Similarly to BPA, prenatal exposure to BADGE showed disturbed development in the amphibian Rhinella Arenarum, including neurological alterations (34). Moreover, in vitro data from cell lines shows increased proliferation after BADGE exposure (26,35,36)
Recently, BADGE has been shown to alter gene expression of cell cycle genes associated with cancer. Developmental exposure of fruit flies to BADGE resulted in overexpression of the genes cyclin B (cycB) and cyclin E (cycE) from the cyclin family (H. Cao, unpublish data). Cyclins are a family of proteins that are involved in the regulation by forming a complex with cyclin-dependent kinases (Cdk) and phosphorylate a number of downstream proteins (37). Cycling E is mainly active during the G1/S-phase of the cell cycle (38) and cyclin B during the mitosis (39). Abnormal expression of cyclins is associated with cancer and overexpression of cyclin E and cyclin B correlates with tumorigenesis (40,41). Cyclin B is involved in growth, migration, metastasis in various cancers (42) and increased levels correlate with poorer overall survival (41).
Additionally, prenatal exposure to BADGE also showed an increased expression of the genes plutonium (plu), Pan gu (png) and giant nuclei/CG5272 protein (gnu). The plutonium protein and pan gu form the serine/threonine-protein kinase PNG that controls the translation of mRNAs, for example, cyclin B (43,44). Giant nuclei forms a subunit of the same PNG kinase complex.
In addition to the alternating in gene expression, Cao et al. also looked at the number of circulating hemocytes in Drosophila larvae. After BADGE exposure, the number of hemocytes was significantly increased compared to control. The increased number of cells could be a result of the alternation in gene expression of cell cycle genes.
To study the fundamental mechanisms of xenobiotics, there is a need for a suitable animal model. Human data from toxicology studies is always limited since it is based on data from mainly epidemiological studies or clinical samplings. Moreover, it is impossible to study how a single toxin interferes with biological functions since we are constantly exposed to a variety of mixtures of environmental xenobiotics. The complex interactions taking place between toxins, endocrine signaling, and neural pathways cannot be easily studied in cell lines. Rodents have frequently been used as a model animal in toxicological studies. However, there are ethical concerns about treating mammals with toxins. Further, rodents have a relatively long generation time, making them time-consuming and costly. Therefore, the fruit fly, Drosophila melanogaster, is an excellent model organism for toxicological studies. D. melanogaster have a short generation time, about ten days, making it an attractive model for studying the long-term effect of xenobiotics. The fly shares several fundamental biological, biochemical, neurological and physiological similarities with mammals. Notably, more than 70% of human disease genes are present as orthologs in D. melanogaster (45,46), making it a valuable model to understand the relationship between the genes and diseases.
In this study, we investigated the effects of BADGE by using D. melanogaster as a model organism. Earlier transcriptome sequencing data revealed overexpression of cycB, cycE, png, gnu and plu after BADGE exposure. Based on the previous findings, we wanted to further investigate the effect of BADGE on gene expression by using quantitative reverse transcription polymerase chain reaction (RT- qPCR) targeting the same genes. Secondly, we used the fly’s hemopoietic system to study cell proliferation after BADGE exposure. To do so, we used a proliferation assay with bromodeoxyuridine (BrdU) staining, a synthetic analog to the nucleoside thymidine. BrdU is incorporated into the DNA during the S-phase of the cell. By visualize BrdU positive cells, it is possible to determine the number of cells undergoing cell division. We hypothesized that BADGE would alter gene expression and as well show an abnormal increase in proliferation of hemocytes, indicating the potential cancerogenous effect of BADGE. The ubiquitous use and exposure make the understanding of how environmental toxins influence the physiology of immediate importance.
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