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Title Assessing the safety of Bisphenol A, its substitutes, and associated detoxification methods using the alternative animal model Caenorhabditis elegans

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Author Chen, Yichang

Publication Date 2018

Peer reviewed|Thesis/dissertation

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Assessing the safety of Bisphenol A, its substitutes, and associated detoxification methods using the alternative animal model Caenorhabditis elegans

A dissertation submitted in partial satisfaction of the requirements for the degree Doctor of Philosophy in Molecular Toxicology

Yichang Chen

2018

ABSTRACT OF THE DISSERTATION

Assessing the safety of Bisphenol A, its substitutes, and associated detoxification methods using the alternative animal model Caenorhabditis elegans

Yichang Chen Doctor of Molecular Toxicology University of California, Los Angeles, 2018 Professor Patrick Allard, Committee Chair

Bisphenol A (BPA) is widely used for producing consumer products such as plastics, receipts, and food packaging. Increased concerns about its safety has led to its replacement with substitutes. However, most of these substitutes share a high degree of structural similarity with

BPA, suggesting that they may possess comparable bioactivity and toxicity. Using the model organism Caenorhabditis elegans (C. elegans ), the consequences on normal reproduction of the BPA substitute bisphenol S (BPS) was investigated. Similar to BPA, BPS resulted in severe reproductive defects, including germline apoptosis and embryonic lethality. However, analysis of meiotic recombination, targeted gene expression, whole transcriptome and ontology analyses as well as ToxCast database analysis indicate that BPS exposure caused reproductive defects are partially achieved via mechanisms distinct from those mediated by BPA. Our findings here not only raise new concerns about the safety of BPA alternatives but also highlight the value of C. elegans as a valuable and relevant model organism for evaluating the toxicity of BPA and its substitutes. Therefore, in the second part of this study, we utilized C. elegans as the model organism to evaluate the detoxification efficiency and safety of a novel

ii enzymatic bisphenol biodegradation system developed by Dr. Shaily Mahendra at UCLA.

Manganese peroxidase (MnP) was utilized in this system to degrade bisphenol compounds

(BPs) in water. Packaging MnP with vault nanoparticles (vMnP) increases its biodegradation efficiency. To answer whether this encapsulation altered enzyme kinetics and resulted in products with different toxicity, a product profile analysis was conducted. Experiments revealed that encapsulation resulted in considerable division in product species and abundance, consistent with the observed changes in the estrogenic activities of BPs. The reproductive toxicity of vMnP-treated samples, as measured in C. elegans , were dramatically reduced for all tested BPs, evidenced by significantly reduced embryonic lethality and germline apoptosis.

Collectively, these results indicate that the vMnP system represents an efficient and effective strategy for the removal of environmental BPs. Finally, although exposure to BPA associates with alterations in hormone production, its effect on mitochondrial cholesterol uptake, the crucial step for hormone biosynthesis, remains unknown. Therefore, the relationship between

BPA and mitochondrial cholesterol transporters was investigated in C. elegans as well. Results indicate that BPA reduced the efficiency of steroidogenic acute regulatory protein (StAR) in cholesterol transport, disturbed intracellular cholesterol homeostasis and resulted in mitochondrial cholesterol deficiency. These changes were positively associated with the reproductive damage observed in worms following BPS exposure, since restoring mitochondrial cholesterol level through exogenous cholesterol supplementation rescued BPA- induced reproductive dysfunctions in nematodes. The findings reported in this study here provide important mechanistic evidence on the effect of BPA on intracellular cholesterol transport and reproduction. They also suggest that the reproductive system of individuals with

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StAR deficiency or low cholesterol level may be sensitive to BPA’s toxicity. In summary, this study not only expands our knowledge of the toxicity of bisphenol compounds but also clearly highlight the value of C. elegans as a powerful in vivo assay model in toxicology research.

The dissertation of Yichang Chen is approved.

Oliver Hankinson

Michael D. Collins

Shaily Mahendra

Patrick Allard, Committee Chair

University of California, Los Angeles

2018

To my parents and my three cats who support me through every hard times of my life

TABLE OF CONTENTS Chapter 1: Introduction------01

References------25

Chapter 2: Exposure to the BPA-substitute Bisphenol S Causes Unique Alterations

of Germline Function------34

Reference------52

Supplementary Materials------55

Contribution------61

Chapter 3: A Vault-encapsulated Enzyme Approach for Efficient Degradation and

Detoxification of Bisphenol A and its Analogues------62

Reference------92

Supplementary Materials------95

Contribution------106

Chapter 4: BPA Antagonizes StAR-dependant Mitochondrial Cholesterol Transport

to Induce Gemline Dysfunction------107

Reference------130

Contribution------135

Chapter 5: Summary and Discussion------136

Reference------143

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LIST OF FIGURES AND TABLES Chapter 1 Introduction

Figure 1.1. Chemical structures of BPA and its substitutes------07

Figure 1.2. Ubiquitous exposure to BPS in humans------10

Figure 1.3. Composition profiles of BPA, BPS, BPF, and BPAP in urine samples------11

Figure 1.4. Vault nanoparticles encapsulation enhances the biodegradation efficiency of manganese peroxidase (MnP) on bisphenol compounds ------16

Figure 1.5. Intracellular cholesterol transport and metabolism------19

Table 1.1. Occurrence of bisphenol analogues in surface water------14

Table 1.2 . Occurrence of bisphenol analogues in sediment and Sludge------14

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Chapter 2 Exposure to the BPA-substitute Bisphenol S Causes Unique Alterations of Germline Function

Figure 1. Bisphenol exposure results in sterility, reduced brood size and increased embryonic lethality in C. elegans ------39

Figure 2. Bisphenols exposure induces germline nuclear loss and apoptosis------41

Figure 3. Bisphenols exposure induces DNA damage checkpoint kinase CHK-1 activation-42

Figure 4. BPS does not alter the turn-over of DSBR protein RAD-51 in late pachytene-----44

Figure 5. Distinct expression changes of genes implicated in DSBR and DNA damage checkpoints activation pathways------45

Figure 6. Bisphenol S exposure impairs homologous chromosome synapsis------46

Figure 7. BPA and BPS show distinct alterations of gene expression at whole transcriptome level------47

Table S1. Bisphenols internal exposure level as measured by gas chromatography-mass spectrometry------55

Table S2. Primer sets used in quantitative RT-PCR experiments------55

Figure S1 . BPA but not BPS exposure results in reduced gonad size------56

Figure S2. Bisphenols exposure induces germ cell loss and apoptosis------57

Figure S3. Bisphenol S exposure impairs chromosome morphogenesis in diakinesis------57

Figure S4. Diameter of mitotic zone nuclei following exposure------58

Figure S5. Concordance between germline nuclear loss and pCHK-1 activation------58

Figure S6. Apoptotic response in spo-11 and DDR mutants------58

Figure S7. Concordance of RNA-seq data with qPCR data------59

Figure S8. Biological similarities of BPA analogues to BPA------59

Figure S9. Assessment of differential expression of genes involved in DNA repair------60

Chapter 3 A Vault-encapsulated Enzyme Approach for Efficient Degradation and Detoxification of Bisphenol A and its Analogues

Figure 1. Enhanced BPA removal by MnP encapsulated in vaults------76

Figure 2. Removal kinetics of BPF (A) and BPAP (B) by MnPs------78

Figure 3. Comparison of product profiles among vMnP and nMnP treatments for BPA, BPF, and BPAP------79

Figure 4. Germline apoptosis of worms exposed to bisphenols and their biodegradation products------83

Figure 5. Embryonic lethality of bisphenols and their biodegradation products------84

Table S1. Recovery of bisphenol parent compounds in concentrated SPE elutes------99

Table S2. Parameters for MZmine------99

Figure S1. Chemical structures of BPA, BPS, BPF, and BPAP------100

Figure S2. Characterization of vault nanoparticles packaged with MnP------100

2+ Figure S3. Enzyme kinetics of vMnP, rMnP, and nMnP using Mn (A), H 2O2 (B), ABTS (C), and guaiacol (D) as substrates------101

Figure S4. No significant BPS degradation in 24 hours------101

Figure S5. Proposed MnP-catalyzed BPA transformation pathway------102

Figure S6. Proposed MnP-catalyzed BPF transformation pathway------103

Figure S7. Proposed MnP-catalyzed BPAP transformation pathway------104

Figure S8. Ethanol vehicle does not affect C. elegans reproductive features------105

Figure S9. Larval lethality of BPs and their degradation products------105

Chapter 4 BPA Antagonizes StAR-dependant Mitochondrial Cholesterol Transport to Induce Gemline Dysfunction

Figure 1. Exogenous cholesterol rescues BPA induced fertility damage------118

Figure 2. Cholesterol prevents BPA induced germline damage------119

Figure 3. BPA disrupts intracellular cholesterol homeostasis------121

Figure 4. StAR is a crucial mediator for BPA to elicit its reproductive toxicity------123

Figure 5. TSPO is not necessary for BPA to elicit its reproductive toxicity------125

LIST OF ABBREVEATIONS

BPA Bisphenol A BPAF Bisphenol AF BPAP Bisphenol AP BPB Bisphenol B BPF Bisphenol F BPS Bisphenol S BPZ Bisphenol Z C. elegans Caenorhabditis elegans CAH Congenital adrenal hyperplasia CHK-1 Checkpoint kinase-1 Cre Creatinine CYP17A1 Cytochrome P450 17A1 CYP19 Cytochrome P450 aromatase DDR DNA damage response E. coli Escherichia coli EDC Endocrine disrupting compounds EDI Estimated daily intakes EEDCs Environmental endocrine disrupting chemicals ELISA Enzyme-linked immunosorbent assay EPA Environmental protection agency FDA Food and drug administration FDR false discovery rate FSH Follicle-stimulating hormone GC-MS Gas chromatography–mass spectrometry GO Gene ontology

IC 50 Half maximal inhibitory concentration IMM Inner mitochondria membrane IVF in vitro fertilization LAL Lysosomal acid lipase LDL Low-density lipoproteins LH Luteinizing hormone Lip Lignin peroxidase LOAEL Lowest observed adverse effect LXR Liver X receptor MnP Manganese peroxidase MVP Major vault protein NGM Nematode growth medium nMnP Natiec form Manganese peroxidase OMM Outer mitochondrial membrane P450scc P450 cholesterol side-chain cleavage enzyme

xii pCHK-1 Phosphorylated checkpoint kinase-1 PCOS Polycystic ovary syndrome qRT-PCR quantitative reverse-transcription PCR rMnP Recombinant MnP-INT RNA-seq RNA sequencing SC Synaptonemal complex Sf9 Spodoptera frugiperda StAR Steroidogenic acute regulatory protein TEM Transmission electron microscopy TR-FRET Time-resolved Förster resonance energy transfer TSPO Translocator protein VDR Vitamin D receptor vMnP Vault-encapsulated MnP 17β-HSD 17β-hydroxysteroid dehydrogenases

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ACKNOWLEDGEMENTS

First and foremost, I would like to thank my inspiration, my mentor and my friend, Dr.

Patrick Allard, for allowing me to join his lab and pursue a career in toxicology, the field that

I have always felt passionate about. He has been such a great mentor working very hard along my side to shape me into the scientist that I am today. I thank him for his patience and great advice throughout the years. He has been nothing but supportive and has allowed me to work independently and run freely with my projects. I am forever grateful for his guidance and help.

Meanwhile, I would also like to thank all the past and present members of the Allard’s laboratory for giving me tremendous amount of help with my research, my life, and my writing.

I cannot achieve what I have now without their help. I would like to specifically thank Jessica

Camacho, Lisa Truong, Rio Barrere-Cain, and Blake Panter, who did tons of proofreading for this dissertation. I would also like to thank all of my committee members, Dr. Hankinson, Dr.

Collins, and Dr. Mahendra for all of their advice throughout the years. They were always willing to meet with me anytime I need help or guidance with my career. I am truly grateful for picking such a great committee.

Also, I would like to thank members of Dr. Shaily Mahendra’s laboratory for the past help they gave to me. Specifically, I would like to thank Meng Wang for designing the experiments and interpretation of data for the bisphenol remediation project. The third chapter of this dissertation wouldn’t exist without his help.

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At the same time, I would also like to thank my family and friends. Everyone has supported me unconditionally and always been there for me when I doubted my life and my career. I could never repay them for the amount of support I have received.

In the end, I would also like to thank UCLA Molecular Screening Shared Resource

Center and Clinical Microarray Core for lending their services that have heavily contributed to my research and specifically, to Dr. Shane Que Hee and Dr. Robert Damoiseaux for training me in gas chromatography-mass spectrometry and Flex station II multi-mode microplate reader. Without them, my research would not be possible. I would like to thank my multiple funding sources including the NIH, SOT, and UCLA. These funds made my doctoral work possible.

Yichang Chen EDUCATION

Peking University, Beijing, China Sept. 2008-Jul. 2010 Master of Science, School of Public Health Peking University, Beijing, China Sept. 2003-Jul. 2008 Bachelor of Medicine (M.D. equivalent), School of Public Health AWARDS

¢ NRSA Institutional Predoctoral Training Grants (T32), UCLA (2016)

¢ Reproductive and Developmental Toxicology Specialty Section Graduate Student Award, 1st Place, 54 th SOT Annual Meeting (2015) st ¢ Poster Session Award, 1 Place, SCCSOT Annual Meeting (2014) th ¢ Best Student Platform Presentation, 45 EMGS Annual Meeting (2014) st ¢ Travel Award, 1 Place, SCCSOT(2013)

¢ Scholarship for Outstanding Student, Peking University (2009) EXPERIENCE Graduate Student Researcher Sept. 2012-Present Molecular Toxicology IDP School of Public Health, University of California, Los Angeles

¢ Project 1 : Exposure to the BPA-Substitute Bisphenol S Causes Unique Alterations of Germline Function . In this study, the reproductive toxicity of bisphenol S (BPS), one of the substitutes of Bisphenol A (BPA) used in plastic industry, was investigated by using the model organism Caenorhabditis elegans (C. elegans ). We found that BPS, similarly to BPA, caused severe reproductive defects including germline apoptosis and embryonic lethality. However, meiotic recombination, targeted gene expression, whole transcriptome, and ontology analyses, as well as ToxCast data mining, all indicate that these effects are mainly achieved via mechanisms distinct from BPA's.

¢ Project 2: Enhanced Removal and Detoxification of Bisphenol A and Its Substitutes by Enzymes Immobilized in Vault Nanoparticles. In this study, we proposed to use vault nanoparticles to stabilize and enhance the manganese peroxidase (MnP) mediated enzymatic reaction to degrade and detoxify bisphenol compounds in the environment. The result showed that vault packaged MnP (vMnP) was able to mediate a remarkably faster and more sustained degradation than its native unpackaged form (nMnP). The reproductive toxicities of bisphenols were also significantly removed by vMnP as treated bisphenols didn’t induce any fertility damage or germline apoptosis in C. elegans.

¢ Project 3: Bisphenol A Exposure Impairs the Transport and Utilization of Mitochondrial Cholesterol to Induce Damages on Reproduction. In this study, whether BPA, as an environmental endocrine disruptor, can impair the steroidogenesis in mitochondria, especially the transport of cholesterol, was investigated with the use of genetic model organism C. elegans .

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Graduate Student Researcher Aug. 2008-Jun. 2010 Department of Toxicology School of Public Health, Peking University

¢ Project 1: Perinatal Bisphenol A Exposure Induces Deleterious Effects on the Reproductive System of SD Rat’s Offspring. The developmental and reproductive toxicity of perinatal Bisphenol A exposure was studied on the rodent animal model. We found that 50 mg/kg BW BPA perinatal exposure could induce morphological changes in reproductive organs, impair the steroidogenesis process, and reduce the testosterone level of exposed offspring.

¢ Project 2: Perinatal Exposure to Bisphenol A Epigenetically Modifies the Expression of Estrogen Receptors. This project was focused on investigating the expression changes of estrogen receptor (ER) α and β after the perinatal Bisphenol A exposure on SD rats’ offspring. The de-methylation happened in the promoter region of both ER α and β, along with their increased transcription and translation, indicating that BPA could epigenetically modify the expression of ER.

Clinical Clerkship Jan. 2006-Jun. 2007 Peking University 9th Hospital, Beijing, China

¢ Rotated among nine departments including the departments of Cardiology, Endocrinology, Pulmonary Medicine, Gastroenterology, Gynecology and Obstetrics, Pediatrics, General Surgery, Orthopedics and Emergency and also participated in medical courses including Internal Medicine, General Surgery, Obstetrics and Gynecology, Pediatrics and other related clinical courses. PUBLICATIONS

¢ Chen Y. , Shu L., Qiu Z., Lee D.Y., Settle J.S., Que Hee S., Telesca D., Yang X. and Allard P., Exposure to the BPA-Substitute Bisphenol S Causes Unique Alterations of Germline Function. PLoS Genet. Jul 29;12(7):e1006223 (2016). 1 1 ¢ Wang M. , Chen, Y. , Kickhoefer V., Rome L., Allard P., Mahendra S., A Vault-packaged Enzyme Approach for Efficient Degradation and Detoxification of Bisphenol A and its Analogues. (First Co-author; In review) (2018)

¢ Chen Y. , Shang L., Wei X., Jiang J., Liu R., Xing L., Wu S., Hao W., The effect of Perinatal Exposure to Bisphenol A in the reproductive system of female offspring rats and changes in the expression of estrogen receptor. Teratogen. Carcin. Mut. 22(1): 165-71 (2010).

¢ Parodi DA., Chen Y. , Sarif J., Allard P., Reproductive toxicity and meiotic dysfunction following exposure to the pesticides Maneb, Diazinon, and Fenarimol. Toxicol. Res. 4:645- 54 (2015).

¢ Ferreira D., Chen Y. and Allard P., Use of the alternative model system C. elegans in developmental and reproductive toxicology. Developmental & Reproductive Toxicology (ed. Dr. Ali Faqi). Springer (2014).

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Chapter 1

Introduction

1.1 Prologue

The fundamental goal of toxicology is to delineate and evaluate the adverse effects of environmental chemicals on biological processes, with a focus on public health risk prevention.

In the United States, there are currently over 80,000 chemicals registered for commercial use, and there is limited human toxicity and exposure data for the majority of these chemicals

(Judson et al. 2009; Liao, Liu, Alomirah, et al. 2012). Toxicity tests with the use of mammalian animal as the model organism are considered the definitive methods and regularly used to establish human exposure guidelines. However, these in vivo tests are both labor-intensive and time-consuming, making it impractical to test all commercially used chemicals for their toxicities. Thus, developing rapid, low-cost in vivo methods with the use of alternative animal model is necessary and important for us to detect potential toxicants in environment.

Removal of pollution or contaminants from the environment can directly reduce human exposure to toxic chemicals and thus prevent their health risk to the public. In addition to environmental toxic chemical treatment, replacing these chemicals with substitutes possessing less ecological hazard profiles is another effective implementation in hazard control. The selection of a substitute can be a very complex process and extreme care must be taken to ensure that one hazard is not being exchanged for another. Therefore, a comprehensive risk assessment should be carried out on the potential substitute before it is put into use.

1.2 Bisphenol A and its health concerns

1.2.1. Bisphenol A and its human exposure

Bisphenol A (4,4’-(propane-2,2-diyl) diphenol, BPA) is an organic synthetic compound first discovered in 1891 by Russian chemist Aleksandr Dianin (Dodds and Lawson 1938). It has been in commercial use since 1957 and incorporated in a wide variety of common consumer products such as plastic bottle, thermal paper, dental composite filling, food packaging and more (Hoekstra and Simoneau 2013). Based on its widespread industrial application and demand, BPA was massively produced and released into the environment during manufacturing processes as well as through leaching from consumer products. This makes it detectable from various environmental matrices, such as indoor dust (Liao, Liu, et al.

2012a), water (Yamazaki et al. 2015a), and food (Liao, Liu, et al. 2012a; Liao and Kannan

2014). Therefore, human can be exposed to BPA via inhalation (Liao, Liu, Guo, et al. 2012), drinking (Jin and Zhu 2016), consuming food (Liao and Kannan 2014), or even receiving receipts from grocery stores (Thayer et al. 2016). Despite its relatively short half-life (6-24 hours) (Volkel et al. 2002), the daily-based long-term environmental exposure makes BPA detectable in various forms of human samples as well, such as urine (2.66 ng/mg Creatinine

(Cre)) (Yamano et al. 2008), saliva (0.2 ng/ml) (Sasaki et al. 2005), breast milk (0.61 ng/ml)

(Sun et al. 2004), colostrum (3.41 ng/ml) (Kuruto-Niwa et al. 2007), ovarian follicular fluid

(2 ng/ml) (Ikezuki et al. 2002), and placenta (103.4 ng/g) (Troisi et al. 2014). Owning to its ubiquitous environmental distribution and its well documented estrogenic activity (Hiroi et al.

1999; Yoshihara et al. 2001; Dodds and Lawson 1938), BPA has become one of the major environmental endocrine disrupting chemicals (EEDCs) and its toxicity has been extensively studied in the past several decades.

1.2.2. Reproductive toxicity of BPA

One of the health concerns associated with BPA is its ability to affect gametogenesis and therefore impair the reproductive function of an organism, which has been discovered from both experimental animal and human based studies. Particularly, sperm count decrease, sperm motility impairment (Richter et al. 2007), and sperm epigenome alteration (Doshi et al. 2012) had been observed in male rodents exposed to BPA under the its estimated lowest observed adverse effect level (LOAEL), the lowest amount or concentration of a chemical to cause its adverse effects. In female rodents, BPA in utero or neonatal exposure was also associated with inhibited germ cell nest breakdown, decreased primordial follicles (Wang, Hafner, and Flaws

2014; Zhang et al. 2012; Zhang et al. 2014), altered follicle type distribution (Gamez et al.

2015), and increased oocyte apoptosis (Zhang et al. 2014). Additionally, increased multi- oocyte follicle number and impaired oocyte meiotic differentiation were also observed in

Rhesus macaque (Hunt et al. 2012) and lamb (Rivera et al. 2011) exposed to BPA, indicating the impact of BPA on folliculogenesis is ubiquitous across different species.

Compared with extensive animal studies from the past, the amount of epidemiology studies that explore the relationship between BPA and its reproductive damage on human is relatively limited. However, a clear negative association was found between urinary BPA concentration and total sperm count, sperm vitality, and sperm motility among male workers with occupational BPA exposure in factories of China whose urinary BPA level (38.7 ng/ml) was 35 times higher than the non- exposure workers in control group (Li et al. 2011). Similar findings were also reported from studies with non-occupational exposure individuals (Lassen

4 et al. 2014) and male partners seeking fertility treatment (Meeker et al. 2010; Knez et al. 2014;

Goldstone et al. 2015). In females, the serum BPA level in infertile women was higher than it in fertile women (Caserta et al. 2013; La Rocca et al. 2014) and associated with recurrent miscarriage (Sugiura-Ogasawara et al. 2005). Further, in women undergoing in vitro fertilization (IVF) treatment, serum or urinary BPA levels were inversely associated with peak estradiol level, number of retrieved oocytes, fertilization rates, and embryo quality, indicating increased BPA exposure may decrease the success rate of IVF treatment (Bloom et al. 2011;

Fujimoto et al. 2011; Ehrlich et al. 2012; Mok-Lin et al. 2010; Souter et al. 2013).

BPA exposure-induced reproductive damages may particularly, but not exclusively, stem from its ability to impair the production of gametes through interfering with germ cell meiosis.

As shown by the existing studies, exposure to a physiologically relevant dose of BPA

(approximately 400 ng/day) in rodents revealed two windows of germ cell sensitivity (Susiarjo et al. 2007b). Exposed adult female mice display chromosome congression failure at metaphase I and II, leading to chromosome segregation errors at anaphase and the generation of aneuploidy oocytes (Hunt et al. 2003). Specifically, the process of synapsis, or the physical linkage of homologous chromosomes during pachytene I, is impaired as revealed by the presence of partially synapsed homologues (Susiarjo et al. 2007b; Allard and Colaiacovo

2010a). Second, the associated process of meiotic recombination is also altered as shown by an increase in homologous crossover frequency (Susiarjo et al. 2007b). The alteration of these two fundamental meiotic processes correlate with an increased incidence of chromosome end- to-end associations as well as oocyte and embryonic aneuploidy (Hunt et al. 2012; Susiarjo et

5 al. 2007b) .

1.3. BPA and its substitute BPS

1.3.1. BPA’s regulation and substitution

Despite BPA’s toxic effects observed in many organisms under experimental conditions, human exposure to BPA is often considered too low to cause deleterious effects (Shelby 2008).

However, in recent years, the description of low and ultra-low dose responses, the identification of new transdermal exposure routes, and the significant amounts of BPA in thermal paper (Geens et al. 2012; vom Saal and Hughes 2005; Welshons, Nagel, and vom Saal

2006) have all renewed the concerns regarding BPA’s safety and provided impetus for its substitution with less toxic alternatives. Because of the health concerns, BPA was first banned in baby bottles manufacturing in Canada in October 2008, followed by its restriction in all food packaging and containers in 2010 (Health Canada 2012, Spetember). The European

Union also banned BPA in baby bottle production in 2011 due to the evidence that early BPA exposure causes precocious puberty (European Commission 2011, October ). In the United

States, BPA has been prohibited in the coating of infant formula packaging since July, 2013

(FDA 2013, July). However, to meet the market demand, several bisphenol analogs that are structurally similar to BPA, such as bisphenol F (4,4'-dihydroxydiphenylmethane, BPF) and bisphenol S (4,4′-Sulfonyldiphenol, BPS) (Fig.1.1), have been introduced as alternatives in industrial applications to manufacture “BPA-Free” products. Most of these analogues were found to display potential ecotoxicities with similar or even stronger estrogenic activity than

BPA (Siracusa et al. 2018). However, whether these BPA-substitutes also cause reproductive toxicity and pose health risks to humans and wildlife is still unclear.

Figure 1.1. Chemical structures of BPA and its substitutes. The common structure of bisphenols is two hydroxyphenyl functionalities connected via a bridging atom, either carbon or sulfur.

1.3.2. The rise of BPS

BPS, originally synthesized as a dye in 1869, is one of the major BPA-substitutes used in manufacturing “BPA-Free” products due to its superior resistance to heat and sunlight

(Glausiusz 2014). It can be discharged into the environment from any part of the production chain and constantly be detected in many environmental specimens nowadays, such as indoor dust, food, and water (Liao, Liu, Guo, et al. 2012; Liao and Kannan 2014; Jin and Zhu 2016).

In addition, BPS has also been widely used in thermal paper as a color developer to replace

BPA, which makes it detectable in cash register receipts as well and expands its human

7 exposure routes (Ndaw et al. 2018; Liao, Liu, and Kannan 2012). Furthermore, aquatic environments, such as lakes and rivers, are often considered as the sites for bioaccumulation and biomagnification of chemical toxicants. The low biodegradability of BPS (Danzl et al.

2009a) makes it accumulate and persist in various types of aquatic environments (Ike et al.

2006; Liao, Liu, et al. 2012a). To date, BPS has become the second biggest contributor to the total amount of bisphenol compounds in surface water, next to BPA and BPF (Yamazaki et al.

2015a; Jin and Zhu 2016). The highest concentration of BPS in surface water was detected in the Adyar River in India, reaching 6.84 μg/ml, at which concentration certain toxic effects can be observed under laboratory conditions (Yamazaki et al. 2015a). In the United States, the occurrence of BPS in the water system is also countrywide. BPS was detected in 76 sludge samples from 74 sewage treatment plants in 35 states collected by the United States

Environmental Protection Agency (EPA) in 2006-2007 with an 86% detection rate and concentrations ranging from 2 ng/g to 1.5 μg/g (Yu et al. 2015). The ubiquity of BPS in the environment suggests a universal human exposure.

The widespread presence of BPS in environment is linked to a near ubiquitous detection in human specimens, such as urine, blood, and breast milk. In 2012, BPS was detected in 81% of 315 urine samples collected from the United States and seven Asian countries, including

China, India, Japan, Korean, Kuwait, Malaysia, and Vietnam (Liao, Liu, Alomirah, et al. 2012).

The highest geometric mean concentration of BPS was found in samples collected from Japan

(1.18 ng/ml or 0.933 μg/Cre), followed by the United States (0.299 ng/ml, 0.304 μg/g Cre) and China (0.226 ng/ml, 0.233 μg/g Cre) (Fig. 1.2). The total exposure level of BPS in the

8 general population of each country was calculated based on the urinary concentrations and it was revealed that the estimated daily intakes (EDI) of BPS in Japan (3.47 μg/day), the United

States (1.48 μg/day), and China (0.71 μg/day) were higher than those of other countries. The relatively high human exposure to BPS in Japan and the United States is in accordance with those countries’ recent efforts to substitute BPA with BPS. Japan has phased out BPA in thermal receipt papers since 2001 and made an effort to develop alternatives, including BPS

(Liao, Liu, Alomirah, et al. 2012). Similarly, in 2006, a large thermal receipt paper producer in the United States announced their replacement of BPA with BPS in paper product manufacturing (Konkel 2013). Furthermore, it is worth noting that, while the concentrations of BPA were always one order of magnitude higher than those of BPS in urinary samples collected from the investigated counties, this situation was reversed in Japan as the urinary concentration of BPS (0.933 μg/Cre) surpassed that of BPA (0.67 μg/g Cre) (Liao, Liu,

Alomirah, et al. 2012). Combined with the fact that the levels and detection frequencies of

BPS in paper products and indoor dust samples in Japan were also higher than those in the other countries (Liao, Liu, and Kannan 2012; Liao, Liu, Guo, et al. 2012), the high urinary

BPS level detected in Japan could be attributed to the early process of substituting BPA with

BPS. This finding suggests that BPS will surpass BPA in a near future to become the predominant bisphenol contaminant as the result of the worldwide BPA substitution movement.

Figure 1.2. Ubiquitous exposure to BPS in humans. A) Creatinine-adjusted geometric mean concentration of BPA and BPS in human urine samples from the United States and seven Asian countries. B) Estimated human exposure doses to BPS in the United States and seven Asian countries, in comparison with exposure doses to BPA. (LaKind and Naiman 2015; Liao, Liu, Alomirah, et al. 2012)

In addition to its wide geographical distribution, human exposure to BPS has also increased throughout time. In a recent study, four major environmental bisphenols, BPA, BPS,

BPF, and BPAF, were measured in 616 urine samples collected from the United States at eight time points from 2000 to 2014 (Ye et al. 2015). While BPS was unable to be detected in the samples collected before 2009, its urinary concentration had progressively increased since

2009 and eventually contributed 25% of the total amount of bisphenols in samples collected in 2014 (Fig. 1.3). In comparison, even though the mean concentration of BPA is always higher than that of other bisphenols in samples collected from the same period, it has gradually declined from 1.3 μg/L to 0.5 μg/L, along with its detection rate decreasing from 98% to 74%.

The change in the occurrence of bisphenols in urine samples reflects their usage history and the movement of BPA replacement in the United States. In concordance with its worldwide

10 distribution, the increase of BPS in human urine samples throughout time suggests BPS, without any further regulation, will soon replace BPA and become the major bisphenol in the environment. Due to the structural similarities between BPA and its substitutes, BPS may possess comparable bioactivity, toxicity, and human health risks to those of BPA.

Figure 1.3. Composition profiles of BPA, BPS, BPF, and BPAP in urine samples. 616 urine samples were collected from U.S. adults at eight time points between 2000 and 2014 (Ye et al. 2015).

1.3.3. The health concerns of BPS

Unfortunately, compared to the number of studies on the toxicity of BPA, the adverse effects of BPS exposure are less investigated and understood. A few of recent studies suggest that BPS, like BPA, may disrupt the function of embryonic neuroendocrine system and further impair the reproductive function of an organism (Qiu et al. 2016; Zalmanova et al. 2017; Ji et al. 2013). For example, adverse effect of BPS on the meiotic maturation process of pig oocytes can be observed at a low exposure level (3 nM) (Zalmanova et al. 2017). Meanwhile, several reproductive studies using Zebrafish ( Danio Rerio ) have reported that BPS is able to affect the development of the hypothalamus, alter expression of steroidogenic genes, and cause hormone imbalance, such as down-regulation in cytochrome P450 17A1 (CYP17A1) and 17β- hydroxysteroid dehydrogenases (17β-HSD), overexpression of cytochrome P450 aromatase

(CYP19a and CYP19b), and increased conversion of testosterone to 17β estradiol (Ji et al.

2013; Naderi, Wong, and Gholami 2014; Cano-Nicolau et al. 2016). Decreased production of gametes, reduced hatching rate, increased occurrence of larval malformation, and growth suspension were also observed in individuals exposed to BPS (Ji et al. 2013; Naderi, Wong, and Gholami 2014). In rodent experiments, exposure to 50 mg/kg.bw/day BPS causes oxidative stress and has anti-androgenic characteristics, evidenced by decreased testosterone level and morphological alterations in testicular tissue (Ullah et al. 2016). However, despite these findings, the toxic mechanisms of BPS for these endpoints, especially gametogenesis, remain elucidated.

In summary, the high degree of similarity between BPA and BPS in both chemical structure and toxicological portrait suggests BPA may not be a suitable BPA substitute. As

BPS is now ubiquitously detected worldwide, a comprehensive risk assessment of BPS is strongly needed. Therefore, the first aim of this study is to identify the reproductive toxicities of BPS and compare them to those of BPA. Understanding the mechanisms by which BPS causes reproductive harm will help us to develop preventive measures to limit the health risks posed by BPS.

1.4. Environmental remediation of Bisphenol compounds

1.4.1. Bisphenols discharged into the environment

Due to its wide-range of application in the industry, approximately eight billion pounds of BPA are produced annually, with more than two billion pounds produced in the United

States (Vandenberg et al. 2012; Corrales et al. 2015). In order to meet the global demand, the

12 annual production of BPA is predicted to grow at an annual rate of 4.6% from 2013 to 2019

(Transparency Market Research 2013). Unavoidably, as one of the massively synthesized chemicals, over one million pounds of BPA are released into the environment annually, mainly through the processing of its manufacture, inefficient removal during wastewater treatment, and leaching from discarded BPA-based materials (Yu et al. 2015). Owning to its substitution,

BPA, along with other bisphenol compounds, have consistently be detected in various ecosystems in recent years (Liao, Liu, et al. 2012a; Liao, Liu, Guo, et al. 2012; Jin and Zhu

2016; Yamazaki et al. 2015a; Yan et al. 2017; Yang et al. 2014; Sun et al. 2017).

Among all environmental sources, the inefficient removal of bisphenols during the wastewater treatment makes the wastewater treatment plant become one of the significant sources for bisphenol compounds to discharge into terrestrial and aquatic environments (Yu et al. 2015; Sun et al. 2017; Lee et al. 2015a). The occurrences of BPA and its substitutes can be detected in all influent, sewage sludge, and effluent of the water treatment process (Table 1.1 and 1.2). In addition, sewage sludge, the organic semi-solid by-product from the wastewater treatment, is often recycled and applied as fertilizer. Its land-application not only offers an inexpensive source of rich nutrients but also releases bisphenols that become sequestered into the terrestrial food chain or leached into groundwater. Since the toxicities of BPA’s substitutes have not been comprehensively studied, improving the remediation efficiency of the wastewater treatment on bisphenol compounds becomes a critical need to reduce their release to environment, as well as their exposure and health risk to publics.

Table 1.1. Occurrence of bisphenol analogues in surface water: Concentration (ng/L, mean or median)

Na: Sample number --: not detected nd: not detectable

Table 1.2. Occurrence of bisphenol analogues in sediment and Sludge: Concentration (ng/g, mean, dry weight)

Na: Sample number --: not detected

nd: not detectable

1.4.2. Biodegradation of bisphenol compounds

The conventional methods applied for removing bisphenols from wastewater include sedimentation, activated carbon adsorption, chemical oxidation, electrochemical techniques, irradiation and others (Bratkovskaja, Vidziunaite, and Kulys 2004). All these methods suffer from serious shortcomings, such as high cost, incompleteness of purification, formation of toxic byproducts, and low efficiency (Bratkovskaja, Vidziunaite, and Kulys 2004). On the contrary, b iodegradation, catalyzed by either whole microbes or free enzymes, has been successfully employed in the treatment of diverse contaminants, such as phenols (Buitron,

Gonzalez, and Lopez-Marin 1998), pesticides (Gavrilescu 2005), and endocrine disruptors

(Hirano et al. 2000a). Compared with the microbial-catalyzed biodegradation, free enzyme mediated biodegradation has many advantages in treating bisphenols in water, such as high specificity to substrates, wide application on various environmental conditions, and ease of process control (Gianfreda et al. 2003). More importantly, it is not constrained by the strict nutrient requirements for microbial growth, nor does it raise public health concerns. In addition, several studies have shown that many fungi generated ligninolytic enzymes, such as manganese peroxidase (MnP), lignin peroxidase (LiP), and laccase, can be utilized in environmental clean-up of various environmental contaminants by oxidative degradation

(Baborova et al. 2006; Gassara et al. 2013). Unfortunately, the implementation of free enzymes is still restricted by their instability in the natural environment due to the nature of their protein-based structures (Tuisel et al. 1990).

1.4.3. Vault nanoparticles encapsulation

Vault cytoplasmic ribonucleoprotein is a cytoplasmic organelle that naturally exists in a variety of eukaryotes and has been proposed to be involved in several essential cellular functions, such as multidrug resistance, transport of the nuclear pore complex, and cell survival (Kedersha et al. 1990). Vault-like nanoparticles can be artificially synthesized and utilized to package desired exogenous components, such as enzymes which can be applied in the biodegradation of bisphenol compounds (Gassara et al. 2013). The encapsulation of MnP with vault nanoparticles could drastically increase the enzymatic stability by protecting packaged enzymes from the external protease degradation and thermal inactivation (Wang et al. 2015a). As an instance, in the previous study, vault packaged Manganese Peroxidase

(vMnP) exhibited a superior stability as well as activity than its native form in degrading BPA and its analog s (Fig. 1.4).

Figure 1.4. Vault nanoparticles encapsulation enhances the biodegradation efficiency of Manganese Peroxidase (MnP) on bisphenol compounds. A) Negative-stained electron micrographs of purified MnP packaged with vault (Wang et al. 2015a). B) Illustration of vault packaging. MnP was incorporated into vaults via fusion to a packaging domain call INT, which strongly interacts with vault’s interior surface. C) Biodegradation efficiency of MnP on bisphenols was significantly enhanced by vault encapsulation in the comparison with recombinant MnP with INT fusion (rMnP) and its native form (nMnP). Above 90% of imputed BPA and BPF, as well as 75% of BPAP were removed after a 24 hours reaction mediated by vMnP, whereas less than 40% of imputed bisphenol compounds were degraded by nMnP.

However, degrading a chemical is not always equal to the success of its detoxification.

The complex structure of ligninolytic enzyme decides its biodegradation system is highly nonspecific. Therefore, treating bisphenol compounds with ligninolytic enzyme, such as MnP, could create metabolites with higher toxicity. Furthermore, while the stability and lifespan of

MnP have been drastically enhanced by the vault nanoparticles package, whether the prolonged reaction could result in different reaction kinetics and metabolomics is still unknown. A metabolite mixture with higher toxicity could be generated through the vMnP mediated reaction than the native form of MnP (nMnP) mediated reaction. A safe bisphenol treatment should reduce or eliminate its toxicity without creating any new toxicant. Therefore, in order to develop the safest remediation process with the least amount of unintended health repercussions, toxicity tests should be employed in the treatment process to evaluate the detoxification efficiency of vMnP mediated degradation of bisphenols, which becomes the second aim of this study.

1.5. BPA and intracellular cholesterol homeostasis

1.5.1. BPA, endocrine system, and reproduction

Though BPA has been gradually replaced with its analogues and began to fade out of our life, a better understanding of its toxicity and related toxic mechanisms will not only quench our thirst for new knowledge but also benefit us to develop a comprehensive risk assessment for evaluating the toxicities of BPA’s substitutes.

Normal reproductive function, such as gamete formation, relies on the fine regulation of neuroendocrine system with hormones as intermediate regulators. Therefore, BPA exposure caused reproductive damages are often associated with its induced endocrine dysfunction.

Specifically, it has been observed from many in vivo studies that exposure to BPA caused alterations in the testosterone and estradiol levels in rodents (Wu et al. 2013; El-Beshbishy,

Aly, and El-Shafey 2013; Xi et al. 2011; Salian, Doshi, and Vanage 2009a, 2009b; Moustafa and Ahmed 2016) and fish (Labadie and Budzinski 2006; Mandich et al. 2007). These changes are often accompanied with fertility impairments such as DNA damage in germ cells (Wu et al. 2013), spermatogenesis abnormalities (Mandich et al. 2007), sperm count and motility decrease (Salian, Doshi, and Vanage 2009a, 2009b), degeneration of ovarian follicles

(Moustafa and Ahmed 2016), and increased post-implantation loss (Salian, Doshi, and Vanage

2009a). Additionally, in human, BPA exposure was correlated with decreased FSH level in

190 sub-fertile men seeking treatment (Meeker, Calafat, and Hauser 2010), pathophysiological change of polycystic ovary syndrome (PCOS), increased LH: FSH ratio, and decreased thyroid stimulating hormone (TSH) in women diagnosed with PCOS (Vahedi et al. 2016).

1.5.2. BPA exposure affects hormone production

BPA induced endocrine imbalance could result from its ability to impair the hormone synthesis. The production of hormones, known as steroidogenesis, follows a complex pathway by which cholesterol is converted to biologically active steroid hormones through a series of reactions mediated by the cytochrome P450 enzyme family (Fig. 1.5). To initiate steroidogenesis, cholesterol stored in the cytosol needs to be transported to the matrix side of

18 the inner mitochondria membrane (IMM), where P450 cholesterol side-chain cleavage enzyme (P450scc; CYP11A1) resides (Farkash, Timberg, and Orly 1986). Cholesterol delivered to the IMM will first be converted to soluble pregnenolone by P450scc and then further transformed to the other steroidal intermediates by enzymes in the mitochondrion and endoplasmic reticulum until the final steroid hormone is synthesized, such as testosterone or estradiol (Miller 1988). Both in vivo and in vitro studies have indicated that BPA can impair the production of progesterone, estradiol, and testosterone through altering the expression of critical genes involved in steroidogenesis, such as Star (Chen et al. 2018; Lee et al. 2013),

Cyp11a1 (Chen et al. 2018; Peretz and Flaws 2013; Medwid, Guan, and Yang 2016), and

Cyp19 (Chu et al. 2018; Lan et al. 2017; Savchuk, Soder, and Svechnikov 2013; Watanabe,

Ohno, and Nakajin 2012).

Figure 1.5. Intracellular cholesterol transport and metabolism. Steroidogenic cells take up circulating low-density lipoproteins (LDL) by receptor mediated endocytosis directing the cholesterol to lysosomes. Free cholesterol can be obtained from either lysosomal acid lipase (LAL) mediated hydrolyzation of cholesterol esters stored in intracellular lipid droplet or de novo synthesis of cholesterol in the endoplasmic reticulum. Free cholesterol reaching the outer mitochondrial membrane requires protein transporter, such as TSPO and StAR, to be delivered to the inner mitochondrial membrane, where is can be converted to pregnenolone by P450scc (CYP11A1) to initiate steroidogenesis process.

It is worth noting that besides the hormone enzymatic biosynthesis process, any error in the uptake, intracellular storage and transport of cholesterol will also affect the production of hormones. However, the impact of BPA on the cholesterol transport, especially the cholesterol mitochondrial internalization, is still largely unknown.

1.5.3. Mitochondrial cholesterol transport The delivery of cholesterol to the IMM is one of the rate-limiting steps in steroidogenesis and its aberrance can result in intracellular cholesterol dyshomeostasis, mitochondrial cholesterol deficiency, and reduction of hormone synthesis (Miller and Bose 2011).

Cholesterol can be transported to the IMM by either non-vesicular transport (protein mediated) or vesicular transport (membrane mediated). In vesicular transport, a vesicle formed from a donor membrane will bud off and then fuse with another membrane to deliver membrane lipids, proteins, and cholesterol between intracellular organelles. In contrast, in the non-vesicular transport, cholesterol requires the facilitation of proteins located on the outer mitochondrial membrane (OMM) to be transported into the IMM (Miller and Bose 2011). These proteins include steroidogenic acute regulatory protein (StAR) and translocator protein (TSPO), which are conserved across species (Fan and Papadopoulos 2013).

StAR is a 37-kDa protein localized on the OMM of mitochondria in steroidogenic cells and implicated as the rate limiting factor in mitochondrial cholesterol uptake (Ikonen 2006;

Bose, Lingappa, and Miller 2002). Although the molecular mechanism of StAR mediated cholesterol transport remains incompletely understood, the indispensable role of StAR in cholesterol mitochondrial translocation was well demonstrated by previous studies in which

20 knocking out StAR resulted in cytosolic cholesterol accumulation, decreased pregnenolone production, and lipoid congenital adrenal hyperplasia (CAH), a severe disorder in which afflicted individuals are unable to synthesize steroids (Caron et al. 1997). A few studies have indicated that BPA exposure can alter the transcription of StAR in mouse Leydig tumor cells

(mLTC-1) (Takamiya, Lambard, and Huhtaniemi 2007) and rat granulosa cells (Samardzija et al. 2018). However, whether such alteration affects the cholesterol transport to mitochondria is still unclear.

TPSO is another cholesterol transporter highly expressed on the mitochondrial OMM.

Early in vivo studies positioned TSPO as a critical component cooperating with StAR to deliver cholesterol into the mitochondria based on the evidence that ligand binding with TSPO increase steroid hormone production (Krueger and Papadopoulos 1990) while inhibiting its expression reduced the steroidogenic capacity of Leydig cell (Boujrad, Hudson, and

Papadopoulos 1993). However, the role of TPSO has become controversial in recent years.

Several in vivo studies showed that normal testosterone production could still be observed in mice with a global knockout of TSPO, along with unaffected testis development, sperm count, and fertility (Tu et al. 2014; Morohaku et al. 2014). Overall, these findings indicate that TSPO is important but not necessary for the mitochondrial cholesterol transport and steroidogenesis.

Furthermore, in contrast with StAR, the effect of BPA on TSPO has not been studied before.

1.5.4. BPA and mitochondrial cholesterol uptake

The effect of BPA on the mitochondrial cholesterol uptake has been rarely investigated.

A recent in vitro study with the use of rat granulosa cell shows that BPA exposure can cause intracellular cholesterol sequestration (Samardzija et al. 2018) and result in decreased hormone production. This finding is in line with the previous reports that BPA exposure results in hepatic lipid accumulation (Marmugi et al. 2012; Ke et al. 2016) and hypercholesterolemia in rats (Marmugi et al. 2014). However, whether this effect of BPA is achieved via affecting the mitochondrial cholesterol uptake is still unknown. Therefore, in order to fill that gap in our knowledge about BPA’s toxic mechanism. The effect of BPA on mitochondrial cholesterol transport will be investigated as the third aim of this study.

1.6. Caenorhabditis elegans as a relevant in vivo animal model for our studies

The comprehensive toxicity of a chemical, especially on reproduction, is difficult to be assessed rapidly by using standard in vivo assays as it requires the use of rodent models over one or two generations. Caenorhabditis elegans (C. elegans ), a free-living nematode, is commonly used in many subfields of biology and toxicology as a model organism since it comes with many advantages over the traditional animal models. As being cultured on agar medium and fed with E. coli , C. elegans can be easily maintained at a low cost. Its short lifespan, which is about twenty days at 20℃, enables the investigation of chronic, even multigenerational, toxic effects of a chemical accomplished in months, instead of years in the comparison with rodent animal models. Additionally, each worm is able to produce approximately three hundred progeny during its entire reproductive period. Its large brood size

22 allows us to quickly identify rare adverse effects of a chemical, such as those related to teratogenicity and mutagenicity.

Though being a self-fertilizing hermaphrodite, C. elegans shares many conserved molecular pathways involved in germ cell maturation processes with humans and other species, enabling it to become an effective animal model for investigating the reproductive toxicity of environmental chemicals. Its reproductive system consists of a symmetrically arranged bio- lobed gonad, in which the process of meiotic differentiation, ovulation and fertilization are spatially and temporally coupled. Furthermore, all stages of meiosis can be identified through the canonical changes in nuclear architecture and morphology inherent to meiosis and are easily visualized by simple nuclear staining technique due to the transparency of C. elegans .

This unique feature allows us to identify the impact of a chemical on each specific meiotic stage of prophase I, such as meiotic recombination, synaptonemal complex (SC) formation, and chromosomes segregation (Allard and Colaiacovo 2010a; Chen, Shu, Qiu, Lee, Settle,

Que Hee, et al. 2016). Moreover, the reproductive toxicity of BPA and its impact on germ cell maturation processes observed from many other species has been successfully verified in C. elegans (Allard and Colaiacovo 2010a). All of these indicate that C. elegans is a suitable animal model for investigating and comparing the reproductive toxicity of BPA and its substitutes.

Estrogenic activity and reproductive toxicity are the signatures of bisphenols. Removing them indicates the success of a treatment on the detoxification of bisphenols. Therefore, all of

23 those bisphenol compounds elicited deleterious effects observed on C. elegans in the first aim of this study could be employed as sensitive biological endpoints for evaluating the safety and efficiency of vMnP mediated biodegradation on removing the reproductive toxicities of bisphenol compounds. An in vitro estrogen receptor binding assay would also be applied to examine the estrogenic activity change of bisphenols through the vMnP and nMnP mediated treatments.

Finally, C. elegans , like humans and many other species, relies on cholesterol derivatives to regulate reproduction and other biological processes. For instance, in C. elegans , cholesterol can be metabolized by DAF-9 (homolog of CYP17A1 in human) to form diachronic acids, which can bind and transactivate orphan nuclear receptor DAF-12 (homolog of human nuclear receptors VDR/LXR) to regulate dauer diapause, reproductive development, fat metabolism, and lifespan (Mukherjee et al. 2017; Motola et al. 2006; Matyash et al. 2004). Depletion of cholesterol can affect the germline mitotic cell cycle progress and consequentially impair worm’s fertility (Merris et al. 2003). Meanwhile, many factors in mitochondrial cholesterol transport, such as the orthologues of TSPO and StAR, have also been identified in C. elegans

(Fan and Papadopoulos 2013). Combining with the fact that C. elegans is unable to synthesize cholesterol de novo and strictly relies on exogenous cholesterol dietary supply (Chitwood and

Lusby 1991), by controlling which the intracellular cholesterol level can be easily manipulated and monitored in experimental conditions, all of these make C. elegans as good model organism to study BPA’s impact on cholesterol homeostasis and mitochondrial cholesterol transport.

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Chapter 2

Exposure to the BPA-Substitute Bisphenol S Causes Unique Alterations of Germline Function

SUPPLYMENTARY MATERIALS

Figure S1

Figure S2

Figure S3

Figure S4 Figure S5

Figure S6

Figure S7

Figure S8

Figure S9

CONTRIBUTION Fertility assessment………………………………………………………….…Yichang Chen

Germline apoptosis assay………………………………………………………Yichang Chen

Germline morphological examination………………………………………….Yichang Chen

RAD-51 time course analysis…………………………………………………..Yichang Chen

CHK-1 immunostaining…………………………………….…………………..Yichang Chen

Synapsis defect analysis…………………..Yichang Chen, Dong Yeon Lee, and Sara J. Settle

Gas chromatography-tandem mass spectrometry analysis…Yichang Chen and Shane Que Hee

RNA-seq analysis…………………………….….……..Yichang Chen, Le Shu, and Xia Yang

RNA extraction and quantitative PCR…………Yichang Chen, Zhiqun Qiu, and Patrick Allard

Biological similarity analysis………………………………………….………..Yichang Chen

Statistical analysis………………………………………Yichang Chen and Donatello Telesca

Chapter 3

A Vault-encapsulated Enzyme Approach for Efficient Degradation and Detoxification of

Bisphenol A and its Analogues

ABSTRACT

We report an effective and environmentally sensitive water treatment approach using enzymes encapsulated in biogenic vault nanoparticles. Manganese peroxidase (MnP), whose stability was remarkably extended by packaging into vaults, rapidly catalyzed the biotransformation of endocrine-disrupting compounds, including bisphenol A (BPA), bisphenol F (BPF), and bisphenol AP (BPAP). The vault-packaged MnP (vMnP) treatment removed 80-95% of each of the tested bisphenols (BPs) at lower enzyme dosage, while free native MnP (nMnP) only resulted in a 19-36% removal, over a 24-hour period. Further product profile analyses revealed that vMnP and nMnP treatments resulted in considerable disparities in product species and abundance, which were consistent with the observed changes in the estrogenic activities of

BPs. To test if vMnP-catalyzed transformation led to the creation of toxic intermediates, we assessed biological hallmarks of BPs toxicity, namely the ability to disrupt reproductive processes and association with estrogen receptors. The toxicity of vMnP-treated samples, as measured in the model organism, Caenorhabditis elegans , was dramatically reduced for all three BPs, including the reproductive indicators of BPA exposure such as reduced fertility and increased germ cell death. Collectively, our results indicate the vMnP system represents an efficient and safe approach for the removal of BPs, and promise the development of vault- encapsulated customized enzymes for treating other targeted organic compounds in contaminated waters.

INTRODUCTION

Enzymatic bioremediation, which utilizes enzymatic catalysis to degrade or transform contaminants in the environment, has been explored for several decades (Franssen et al. 2013;

Hirano et al. 2000b; Mir-Tutusaus et al. 2018; Wei et al. 2013). Benefiting from high efficiency and specificity of enzymes, enzymatic treatment is therefore considered effective and, because of its reduced chemical and energy use, congruent with the principles of environmental sustainability (Cipolatti et al. 2016; Franssen et al. 2013; Wei et al. 2013). However, as many enzymes are not stable outside living cells, and can be inactivated by heat, co-contaminants, and products formed by their own activities, the use of enzymes in water treatment requires high dosage and frequent replenishment, which raises cost and limits their applications in large scale systems (Franssen et al. 2013). A potential solution is to entrap enzymes in solid supports, such as alginate beads, hollow fiber membranes, and magnetic particles (Franssen et al. 2013), to lower dosage and prevent inactivation, thus reduce the cost. But entrapped enzymes usually become less active due to substrate diffusion resistance caused by the solid supports (Cipolatti et al. 2016; Franssen et al. 2013; Meryam Sardar 2015). Recent advances in nanotechnology have provided a wide variety of nanomaterials that are potential alternatives to conventional entrapment supports. Owing to their small dimensions, substrate diffusion problems can be minimized in nano-immoblization, which benefit enzyme catalytic efficiency (Cipolatti et al.

2016; Meryam Sardar 2015). However, most of these materials, such as carbon nanotubes, polymeric nanoparticles, and mesoporous metal oxides, require harsh synthesis conditions and generate hazardous wastes (Meryam Sardar 2015).

The use of bio-nanomaterials in enzyme stabilization is gaining more attention, as they are synthesized under physiological conditions and create less waste streams (Polka, Hays, and Silver 2016). We recently reported a protein scaffold based stabilization approach by encapsulating enzymes in vault nanoparticles (Wang et al. 2015b). Vaults are the largest natural ribonucleoprotein particle with dimensions of 41 x 41 x 72.5 nm, which are synthesized in humans and many other eukaryotes (Rome and Kickhoefer 2013). Each native vault consists of 78 copies of major vault protein (MVP), which assemble into the outer shell of the particle, and several copies of two different vault-associated proteins and small, untranslated RNAs (Rome and Kickhoefer 2013). Synthesized from heterologously expressed

MVP in insect cells, recombinant vaults are empty protein shells that are morphologically identical to native vault particles, and do not cause any adverse public health effects (Han et al. 2011). Recombinant vaults have a core cavity of around 3.87 x 10 4 nm 3 (Stephen et al.

2001), which enable it to be a plausible carrier for encapsulating large macromolecules. The

INT domain, a protein sequence that was derived from one of the vault-associated proteins, has a strong non-covalent interaction with vault interior binding sites, and can direct and sequester fusion proteins containing this domain inside vaults (Kickhoefer et al. 2005). Using the INT fusion strategy, manganese peroxidase (MnP) was encapsulated in vaults, and the resulted vault-encapsulated MnP exhibited better stability during storage and higher resistance against heat inactivation than free MnP (Wang et al. 2015b). However, the effectiveness of vault encapsulation towards enhancing enzymatic treatment of various water contaminants is still unknown.

Bisphenol A (BPA), a chemical commonly used in plastic, paper, and food packaging industries, is one of the most prevalent endocrine disrupting compounds (EDC) in the environment (Liao, Liu, et al. 2012b). A large number of studies have epidemiologically and mechanistically linked BPA exposure to a variety of significant adverse health effects including, most notably, a strong impact on reproduction and fertility (Chen, Ike, and Fujita

2002). In recent years, these health risk concerns associated with its exposure have led to a substitution of BPA with structural analogues, such as bisphenol F (BPF), bisphenol S (BPS) and bisphenol AP (BPAP) (Figure S1). Such analogues are now being used in numerous commercial products and, consequently, they are now also found in different water systems, such as surface water (Yamazaki et al. 2015b), wastewater (Lee et al. 2015b), and sediments

(Liao, Liu, et al. 2012b). However, due to their high degree of structural similarities with BPA, many of these substitutes have been shown to possess similar endocrine disrupting activity and reproductive toxicity as BPA (Chen, Shu, Qiu, Lee, Settle, Hee, et al. 2016; Audebert et al. 2011).

Several oxidative enzymes, such MnP, horseradish peroxidase, and laccase, have been demonstrated to mediate transformation of BPA via coupling reactions or scission reactions

(Huang and Weber 2005; Hirano et al. 2000b). However, similar to many other oxidation processes, enzymatic catalysis does not completely mineralize bisphenols (BPs), leaving a wide range of intermediates (Hirano et al. 2000b; Lu et al. 2013; Olmez-Hanci, Arslan-Alaton, and Genc 2013). Since their chemical structure is similar to that of their parent compounds, such intermediates are also likely to pose health risks, especially the reproductive effects,

66 which are considered as hallmarks of BPs’ toxicity (Allard and Colaiacovo 2010b; Chen et al.

2017; Chen, Shu, Qiu, Lee, Settle, Hee, et al. 2016; Hunt et al. 2003; Kato et al. 2006; Lind and Lind 2011; Susiarjo et al. 2007a). Several studies have assessed toxicity of BPA oxidative transformation products, but focused on acute toxicity only (Lu et al. 2013; Olmez-Hanci,

Arslan-Alaton, and Genc 2013). Evaluation of reproductive toxicity of such intermediates will provide deeper understandings of detoxification processes of BPs and benefit efficiency assessment of various remediation strategies.

The aim of this study was to develop a vault encapsulated enzymatic system as an effective and sustainable approach towards contaminants removal and detoxification. First, the effect of vault encapsulation on MnP enzyme kinetics was assessed. Then, we investigated the transformation of BPA and its analogues BPS, BPF, and BPAP by vault-encapsulated MnP

(hereafter vMnP) at low enzyme dosage, and compared it with unencapsulated MnPs. Finally, we used a combination of in vitro and in vivo assays to assess whether the degradation of the parent compound led to the production of transformation products that caused lower estrogenic and reproductive effects.

MATERIAL AND METHODS

Preparation of Vault Nanoparticles Encapsulated With MnP

Vaults and recombinant MnP-INT (rMnP) were expressed in Spodoptera frugiperda (Sf9) insect cells infected with baculoviruses encoding either hMVP or rMnP as previously described (Wang et al. 2015b). The rMnP was encapsulated in vaults and purified following standard vault processing protocols (Wang et al. 2015b; Kar et al. 2011). Purified vMnP was fractionated on a 4-15% SDS-PAGE gel and analyzed by Coomassie staining and Western blotting using primary rabbit anti-INT antibody and secondary goat anti-rabbit IgG (H+L)

(IRDye 800CW, LI-COR). Negative-stain Transmission Electron Microscopy (TEM) was then performed to confirm the intactness, shape, and size of encapsulated vault particles.

Number-based particle size distributions and zeta potentials of empty and encapsulated vaults were determined using PALS (ZetaPALS, Brookhaven Instruments).

Enzymatic Kinetics Studies

Concentrations of non-encapsulated rMnP or vMnP were determined by enzyme-linked immunosorbent assay (ELISA). Antibodies for ELISA were primary rabbit anti-INT antibody and secondary goat anti-rabbit IgG HRP antibody (BioRad), which were quantified by using the chromogenic reaction of TMB (Dako) according to the manufacturer’s instructions. Native

MnP (nMnP) was purified from Phanerochaete chrysosporium culture as described previously

(Wang et al. 2015b), and quantified by measuring its absorbance at 406 nm which is maximum

-1 -1 absorption wavelength of native MnP enzyme ( ε406nm = 129.3 mM cm ) (Wariishi,

Akileswaran, and Gold 1988).

Manganese divalent ion substrate assays were performed in 200 μL mixture containing pH 4.0 50 mM malonate buffer, MnP, MnCl 2 (50–1000 μM), and 100 μM H 2O2. Assays for

H2O2 substrate were performed in the similar system but containing 1000 μM MnCl 2 and 5–

100 μM H 2O2. For the substrates ABTS (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid)) and guaiacol, the assays were carried in 50 mM malonate buffer containing MnP enzyme, 2 mM MnCl 2, 100 μM H 2O2 with either ABTS (2.5–500 μM) or guaiacol (5–125 μM)

(details in SI). All kinetics assays were carried out at room temperature in triplicate. Initial reaction rates of each substrate were plotted against corresponding concentrations, which were fit to Michaelis-Menten kinetics model using non-linear regression.

Transformation of BPs

All BPs removal reactions were performed at 25°C in a shaking incubator (250 rpm). For each BP, the reaction mixture was composed of 80.3 μL of pH 4.5 50 mM malonate buffer, 10

μL of each type of MnP, 0.74 μL of 20 mM BP dissolved in methanol, 7.5 μL of 20 mM MnCl2 and 1.5 μL of 20 mM H 2O2, with a final volume of 100 μL. MnP enzyme was dosed at 19.3,

15.3, 23.3, and 23.3 U/L for BPA, BPS, BPF, and BPAP reactions, respectively. The enzyme activity assays were carried out in a pH 4.5 50 mM malonate buffer containing 2 mM MnCl 2,

400 μM H 2O2, and 100 μM ABTS. Absorbance increases at 420 nm were recorded for 30 seconds with a 2-second interval for calculating initial ABTS oxidation rates. Enzyme free conditions were included to correct for any non-enzymatic losses of BPs. At each pre- specified time point, triplicate samples were terminated by adding three volumes of methanol,

69 followed by filtration through 0.2 μm syringe filters. The residual BP concentrations were measured using a HPLC as described in the SI. BPA removal was also tested at pH values ranging from 4.0 to 5.5 with a gradient of 0.5. Reaction mixtures were set up as described above in 50 mM malonate buffers at various pHs, each with an initial enzyme activity of 15.3

U/L. Samples were collected at 0 and 24 hours in triplicates, and analyzed using HPLC.

Characterization of Products

Eleven-milliliter reactions in pH 4.5 50 mM malonate buffers, containing 4.97 x 10 -2 μM

MnP (vMnP or nMnP), 1.5 mM MnCl 2, 300 μM H 2O2, and 150 μM BP (BPA, BPF, or BPAP), were incubated at 25 °C, 250 rpm. Matrix tests containing all components except for MnP and

BPs were performed as background negative controls, and enzyme-free assays containing all components except for MnP were performed as positive controls. Recoveries of BPA, BPF, and BPAP were also evaluated in the system only contained malonate buffer and 150 µM of

BP (Table S1). After 24-hour reaction, 1 mL of solution was collected from each sample and mixed with 2 mL of methanol, and stored prior to determining residual BP concentrations using HPLC. The remaining 10 mL solution was subjected to solid-phase extraction (SPE) and processed as described in SI. The final concentrated SPE eluate in 10 µL ethanol was used for product characterization and toxicity tests. Two microliters of final concentrated SPE eluate were diluted in 50 μL of methanol, and the diluted samples were then subjected to

UPLC/MS analyses to characterize products (details in SI). Qualitative and quantitative analyses of UPLC/MS data were carried out using MZmine 2. Detailed data processing methods and parameters are listed in Table S2.

Estrogen receptor competitive binding assay

The binding affinities of each BP and its transformation products towards estrogen receptors α and β were examined by ThermoFisher Scientific performing a time-resolved

Förster resonance energy transfer (TR-FRET) based ER competitive binding assay according to the manufacturer’s instructions. Briefly, BPs or their metabolites compete with a fluorescent

ER ligand (tracer) for binding to the human ER α or β. The displacement of the tracer from the ER reduces fluorescent signal emission triggered by the excitation of conjugated terbium on the receptor. Based on the fluorescence loss, a dose-response curve was generated for each tested chemicals and the half maximal inhibitory concentration (IC 50 ) was then calculated.

Nematode chemical exposure

Caenorhabditis elegans nematodes were exposed to numbered and blinded samples of

BPA, BPF, and BPAP as well as their degradation products following the protocol described previously (Allard et al. 2013). Briefly, worm embryos were collected from the sensitized strain carrying the yIs34[ Pxol-1::GFP, rol-6] reporter construct (Allard et al. 2013) by hypochlorite sodium treatment followed by L1 synchronization. L4 stage larvae were then transferred to M9 liquid culture buffer mixed with 100 µM of each BP or their degradation products for 24 hours for germline apoptosis assay (peak of germline morphology and function), and 48 hours for fertility assessment (to maximize the number of impacted germ cells becoming embryos). Worms were cultured with heat-inactivated bacteria as food in order to avoid potential bacterial metabolism of the compounds. New concentrated heat-inactivated

71 bacteria were added at 24 hours when performing a 48 hour exposure to prevent food depletion.

Germline apoptosis assay

After 24 hours exposure as described above, worms were incubated with 25 µg/mL of acridine orange in M9 solution at room temperature for 2 hours to stain the apoptotic nuclei in the germline as described previously (Gartner, Boag, and Blackwell 2008). After staining, the worms were transferred to new NGM plates for a 30 mins recovery and healthy worms were selected for microscopic examination. The total number of apoptotic nuclei in the posterior gonad of each worm was counted by fluorescence microscopy.

Fertility assessment

After 48 hours exposure, worms from each treatment group were individually transferred to new NGM plates without cholesterol and transferred every 12 hours to a new plate. The numbers of eggs laid by each worm, larvae hatched from these eggs (i.e. embryonic lethality), and larvae successfully reaching adulthood (i.e. larval lethality) were tallied in the three days after exposure.

RESULTS

Characterization of vMnP

We first assessed whether MnP and vault complexes were properly formed in vitro by

Western blot analysis, Coomassie staining, and TEM images (Figure S2A and S2B). Vaults containing rMnP exhibited similar morphology and zeta potentials as empty vaults, but showed a slightly up-shifted distribution of hydrodynamic diameter (Figure S2C and S2D,

Details in SI).

Previously, we found that encapsulation in vaults elevated the apparent Michaelis-Menten half saturation constants (K m) for ABTS (Wang et al. 2015b), but the mechanism was not established. To fully understand the effect of vault encapsulation on MnP kinetics, we further

2+ evaluated K m and turnover numbers ( kcat ) of four substrates including Mn , H 2O2, ABTS, and guaiacol. Results are summarized in Table 1, and the plots are shown in Figure S3. For all substrates under study, K m values of recombinant rMnP ranged from 30% higher for guaiacol

(80±15 vs. 61±7µM) to 17% lower for ABTS (75±3 vs. 90±6 µM) than the numbers for nMnP, indicating that heterologous expression in Sf9 cells and fusion of INT domain did not substantially alter the affinity between the MnP enzyme and its substrates. Comparing K m values between rMnP and vMnP, no significant change was observed for H 2O2 and guaiacol, but a decrease and increase were noted for Mn 2+ and ABTS respectively. It is also observed that rMnP displayed about 3-fold lower kcat relative to the nMnP for each of the substrates tested, which is commonly found for recombinant enzymes (Chen, Zaro, and Shen 2013).

When comparing kcat of vMnP and rMnP, the changes ranged from about 20% decrease for

Mn 2+ (20.3±0.5 vs. 26.4±0.5 s -1, respectively) to no significant change in the case of guaiacol

-1 (13.3±1.2 vs. 14.0±1.3 s , respectively). The changes in overall catalytic efficiency ( kcat /K m) followed similar pattern as kcat . Native MnP showed about 3-fold higher efficiency than encapsulated or free INT-fused MnP for all four tested substrates, which is most likely attributed to its higher kcat numbers. Comparing the catalytic efficiencies of vMnP and rMnP, these two MnPs showed close kcat /K m values (less than 10% difference) for the substrates studied in this experiment except for ABTS. The ABTS kcat /K m value of rMnP was about 1.6- fold higher than that of vMnP, which mostly resulted from the lower ABTS K m value for rMnP.

Together, these experiments confirm that encapsulation in vault particles has limited effect on the enzyme’s kinetic properties.

Transformation of BPA

Several 24-hour time-course removal tests were performed using vMnP, rMnP, and nMnP, each with a low initial enzyme dosage at 19.3 U/L, which is 10-100 times lower than the dosage employed in previous peroxidase-catalyzed degradation studies (Huang and Weber

2005; Hirano et al. 2000b). As shown in Figure 1A, BPA concentration quickly dropped by

20-38% in the first 30 minutes. Afterwards, nMnP and rMnP mediated BPA conversion stopped, and no further significant removal was observed. In contrast, BPA transformation under vMnP catalysis lasted for at least another 4 hours. After 24 hours, vMnP achieved approximately 96% removal of BPA, whereas only 42% and 25% BPA removals were observed for rMnP and nMnP, respectively (Figure 1A).

BPA conversion mediated by vMnP followed a pseudo-first order kinetics for the first 4 hours (Figure 1B), indicating there was no enzyme activity loss during this period. Between

4 and 24 hours, the concentration of BPA further decreased from 10% to 4% of the initial concentration, indicating that vMnP still maintained activity. However, reactions no longer followed the pseudo-first order kinetics. The alteration of BPA transformation kinetics was probably due to the competitive inhibition of vMnP by products formed by its activities, as over 90% of BPA was converted after 4 hours and the resulting products were also favorable substrates of MnP enzymes (Hirano et al. 2000b). For rMnP and nMnP, the pseudo-first order conversion of BPA was only maintained for 30 minutes or less (Figure 1B). Figure 1C shows the change of nMnP mediated BPA conversion rates, which are calculated by normalizing concentration decreases to the intervals between two time points over the 24-hour testing period. The rate peaked in the first ten minutes to 113.0 μM/h, and then rapidly decreased to near-zero in 2 hours. Similar results were also observed for rMnP, suggesting that both unencapsulated enzymes quickly lost their activities after initiating reactions, and had significantly shorter lives under experimental conditions.

Next, we compared the performance of the three MnPs for removing BPA at different pHs in 24 hours (Figure 1D). The results indicate that vMnP consistently showed better performance than unencapsulated rMnP and nMnP at pH 4.0, 4.5, and 5.0. However, due to fact that optimum pH for MnP is around 4, very low BPA removal was observed at pH 5.5.

Therefore, these results suggest that vMnP has longer functional longevity than nMnP, and can still efficiently remove BPA at low enzyme dosage.

Figure 1. Enhanced BPA Removal by MnP Encapsulated in Vaults. (A) Removal of BPA Mediated by vMnP, rMnP and nMnP. In 24 hours, vMnP degraded nearly 100% BPA, as compared with only 30-40% BPA degradation by rMnP and nMnP enzymes. (B) Pseudo- first order kinetics of BPA transformation. vMnP mediated BPA transformation followed pseudo-first order kinetics in the first four hours, but nMnP and rMnP driven BPA conversion only obeyed the kinetics for the first 30 minutes. (C) nMnP mediated BPA conversion rates. The rate of nMnP catalyzed BPA removal immediately decreased after initiating reaction. (D) Vault-packaging Enhanced BPA Removal at Various pHs. Error bars represent one standard error of the mean (n=3).

Transformation of BPA Analogues

Although BPA has been progressively replaced away from some commercial products, most of its substitutes are analogues that share a high degree of structural similarities, suggesting these alternatives may also be treatable by peroxidases. Therefore, we evaluated the effectiveness of vMnP for removing three widely used BPA analogues including BPS, BPF, and BPAP (Liao, Liu, et al. 2012b).

Significant BPF and BPAP degradation was observed in the presence of each type of MnP tested, with BPF following very similar degradation kinetics compared to BPA (Figure 2A).

After 24 hours of reaction, the concentration of BPF decreased to 10.4% of its initial concentration for the vMnP treatment, while it only reached 53.7% and 64.4% after rMnP and nMnP treatments, respectively. For unencapsulated MnPs, most of the BPF removal occurred in the first hour and no further concentration decrease was seen between 1 and 24 hours, while vMnP-mediated BPF conversion increased from 50.2% to 64.8% between 1 and 3 hours and finally reached 89.6% at 24 hours. In the case of BPAP, only 19.0% degradation occurred by nMnP treatment, which is much lower than that of BPA and BPF, the rMnP mediated BPAP degradation reached 85.1% in 24 hours (Figure 2B). Interestingly, vMnP exhibited slower

BPAP conversion than rMnP in the first 6 hours. About 81.0% BPAP removal was observed for rMnP after 6-hour reaction, while only 43.5% BPAP degradation was observed for vMnP.

Due to its low solubility, BPAP forms small aggregates when added into reaction solutions.

These aggregates have larger size than the space between MVP peptides forming vaults’ shell and have limited diffusion, thus the concentration of BPAP inside of the vaults is lower than when in solution and very limited amount of BPAP is accessible to vMnP. Consistent with the lower diffusion of BPAP, we observed that over longer incubation period (beyond 6 hours)

74.7% removal was achieved. For rMnP, the degradation rate only increased by 4.1% between

6 and 24 hours, indicating most of the enzyme lost its activity after 6-hour reaction. BPS was not degraded by all three MnPs within 24 hours (Figure S4). Thus, the results of BPF and

BPAP degradation further demonstrate that encapsulation of MnP in vaults significantly extends enzyme life in reactions and improves contaminant transformation efficiency.

Figure 2. Removal Kinetics of BPF (A) and BPAP (B) by MnPs. For BPF, vMnP catalysis reached nearly 90% removal, while rMnP and nMnP only achieved 46% and 35% removal, respectively. In the case of BPAP, vMnP showed lower removal than rMnP in the first 6 hours, but eventually caught up at 24 hours. Error bars represent one standard error of the mean (n=3).

Enzymatic Transformation Product Profiles

Since vMnP and rMnP exhibited different degradation kinetics, we next characterized and compared the product profiles after vMnP and nMnP treatments for each BP using UPLC/MS.

Products were separated by their retention times and mass to charge ratios (Figure 3). This preliminary analysis indicates that vMnP and nMnP treatments generated significantly different product species as the reactions progressed. Referring to previously reported mechanisms of peroxidase catalyzed BPs transformations (Huang and Weber 2005; Hirano et al. 2000b), the proposed MnP mediated reaction pathways for BPA, BPF, and BPAP are

78 presented in Figure S5, S6, and S7, respectively. In general, the parent BPs undergo scission reactions that breakdown BPs to small-mass products, followed by coupling reactions that generate oligomeric BPs.

Figure 3. Comparison of Product Profiles Among vMnP and nMnP Treatments for BPA, BPF, and BPAP. Products were separated and named by their UPLC retention time and mass-to-charge ratios, and plotted based on their relative abundance. Treatments by vMnP and nMnP generated significant different products. Grey color indicates that the ion was not detected in the sample.

In the case of BPA, 17 and 24 products were detected after vMnP and nMnP treatments respectively, most of which were not found in proposed MnP mediated BPA transformation.

The packaged and unpackaged forms of MnP enzyme shared only 4 common products.

Among the 17 species identified for vMnP treatment, 4 (Figure S5, species A1-A4) were

79 generated from proposed MnP catalysis, making up about 43.6% of total MS response. For nMnP treatment, 24 species were identified, two of which were from the MnP catalyzed reaction (Figure S5, species A1 and A3), and accounted for only 9.1% of total MS response.

The ion 133, which is the first intermediate in the proposed pathway, was reported at an abundance of 13.1% of total MS response for vMnP as opposed to only 3.3% for nMnP. The next species in the pathway (Figure S5, species A2) was only found in the sample from vMnP treatment. BPA trimers or oligomers (Figure S5, species A4), which are formed at the end of the proposed pathway, exhibited an abundance of 10.1% of total MS response for vMnP, while those were not detected in the samples from rMnP treatment.

For BPF (Figure 3, middle panel), vMnP and nMnP treatments yielded more similar product profiles. Sixteen species were detected in the sample from vMnP treatment, five of which (Figure S6, species F1- F5) were from proposed MnP-mediated reactions and made up for about 81.0% of total MS response. For nMnP treatment, 23 species were identified, which shared 7 species with the vMnP treatment, four of which (Figure S6, species F1, F2, F3, and

F4) were found in proposed enzymatic pathways and were about 60.0% of total MS response.

Product F2 (4-hydroxybenzaldehyde), which is the second product in MnP catalyzed BPF transformation pathways, was the most abundant species in both treatments, suggesting the subsequent reactions converting F2 may not be favored. Although species F1 and F2 were very abundant in both conditions, they still exhibited a higher percentage in the sample from vMnP treatment.

Finally, the product profiles of BPAP were more complex than those of BPA and BPF

(Figure 3, right panel). Twenty-nine and thirty-two product species were identified for vMnP and nMnP treatments respectively, only 6 of which were shared between two enzymes. Five species (Figure S7, P1-P5) in the proposed enzymatic pathway were found from vMnP treatment, which accounted for about 33.8% of the total MS response, while only 3 were identified from nMnP treatment, making up for less than 8% of total MS response, which agree with the findings for BPA and BPF. Together, these results imply that treatments by vMnP and nMnP resulted in significantly different product profiles, and transformation of BPs by vMnP fitted with proposed MnP-mediated pathways better than the transformation by nMnP.

Reduction in BPs’ Toxicity

Exposure to BPA has been associated with a variety of toxic responses including strong reproductive effects across a variety of organisms through mechanisms that are sometimes distinct from its weak affinity for the estrogen receptor (ER) (Maffini et al. 2006; Rubin 2011;

Vandenberg et al. 2007). BPA’s reproductive effects can be considered a hallmark of its toxicity as BPA exposure leads to a decrease in fertility that correlates with a decreased viability of germ cells in a great number of animal species examined to date, including humans and well-established laboratory model organisms such as mouse, rat, zebrafish, drosophila, and C. elegans worms (Hunt et al. 2003; Susiarjo et al. 2007a; Kato et al. 2006; Lind and Lind

2011; Chen et al. 2017; Atli 2013; Allard and Colaiacovo 2010b). The mechanisms underlying

BPA’s reproductive effects are also well conserved as BPA exposure was shown to cause an

81 increase in germ cell death by apoptosis and an increase in chromosome errors and lethality in mouse and C. elegans early embryos (Hunt et al. 2003; Susiarjo et al. 2007a; Allard and

Colaiacovo 2010b). In C. elegans , these findings were extended to the BPA analogue BPS suggesting that the similarity in chemical structure imparts comparable effects on germ cells

(Chen, Shu, Qiu, Lee, Settle, Hee, et al. 2016). The remarkable conservation of reproductive toxicity outcomes caused by BPA exposure was leveraged here to examine whether MnP- mediated degradation of BPA and its analogues decreases their associated toxicity by monitoring the model organism C. elegans .

Following a single-blind protocol, we first examined the induction of germline apoptosis in the midpoint of the C. elegans gonad by acridine orange staining. Following exposure to

BPA and BPF at the reference concentration of 100µM for 24 hours spanning the onset of reproduction (L4 to adult), we observed a significant 40% to 60% increase in the number of apoptotic nuclei when compared to the 0.1% ethanol vehicle control (P≤0.05, Student's t-test)

(Figure 4). A similar significant increase was observed in the enzyme-free/mock treatment group. In contrast, vMnP treatment dramatically reduced BPA, BPF, and BPAP -mediated germline apoptosis effect to levels indistinguishable from controls. The results were more variable for the nMnP as BPA and BPF degradation did not reduce their impacts on germline apoptosis while it did for the BPAP group.

Figure 4. Germline Apoptosis of Worms Exposed to Bisphenols and Their Biodegradation Products. As compared to vehicle control (0.1% ethanol), 24 hours exposure to 100 μM of BPA (A), BPF (B), or BPAP (C) significantly increased apoptotic germline nuclei numbers by 66%, 64% and 38% respectively. The increase in germline apoptosis was also observed in the mock bisphenols groups with no treatment (Enzyme Free), as well as the nMnP treated BPA and BPF groups. In contrast, biodegradation mediated by vMnP completely removed the ability of BPA, BPF, and BPAP to induce germline apoptosis compared to the Matrix control (no chemical). Student’s t-test performed for the comparison between the control and each 100 μM bisphenol group. One way- ANOVA with post-hoc Tukey HSD test performed for the comparison among all bisphenols treatment groups. *: P<0.05 and **: P<0.01. N=4.

Next, we assessed the impact of the various treatments on BPA, BPF, and BPAP-induced embryonic lethality (Figure 5). Compared to control, both BPA and BPAP exposure significantly increased the embryonic lethality by 48% and 38% respectively (P≤0.05, student's t-test) while BPF exposure induced a 19% increase but failed to reach a statistical significance. Among all treatment groups -enzyme free/mock, nMnP, and vMnP- only the latter led to a reduction in embryonic lethality to a level comparable to controls. The stronger reduction in germline apoptosis compared to embryonic lethality can be explained by the fact

83 that the solvent used 0.1% ethanol alone causes some degree of embryonic lethality and therefore increased the baseline of the lethality assay (Figure S8). However, the embryonic results are particularly significant considering that no effect of any of the BPs was detected on later stages of the nematode’s development as measured by larval survival assay (Figure

S9).

Figure 5. Embryonic Lethality of Bisphenols and Their Biodegradation Products. Embryonic lethality is calculated as the percentage of unhatched eggs in all the eggs laid by each worm. Compared with the vehicle control (0.1% ethanol), exposure to 100 μM BPA (A) or BPAP (C) significantly increases the embryonic lethality by 48% and 38% respectively, while 100 μM BPF (B) showed a 22% increase but failed to reach statistical significance. After the biodegradation reaction, vMnP, but not nMnP, bisphenols treatment led to a reduction in embryonic lethality to a level comparable to the buffer control group (Matrix). Student’s t-test performed for the comparison between the control and each 100 μM bisphenol group. One way-ANOVA with post-hoc Tukey HSD test performed for the comparison among all bisphenols treatment groups. *: P<0.05. N=10-13.

The dramatic decrease in the BPs’ reproductive toxicity after vMnP treatment could be due to a reduction in their estrogenic activity. To test this possibility, we assessed the ability

84 of nMnP and vMnP to decrease the association of BPA, BPF, and BPAP with the estrogen receptors α and β following treatment using a fluorescence displacement assay. After 24 hours, both nMnP- and vMnP-mediated transformation products showed a decreased ability to bind the ERs for all tested BPs (Figure S10). Interestingly, vMnP exhibited a better performance towards reducing BPAP ER binding while nMnP displayed a preference towards BPA and BPF.

However, taken together, these results highlight the potent detoxification of BPA, BPF, and

BPAP by vMnP.

DISCUSSION

Application of white-rot fungi for removing organic micro-contaminants in water is becoming increasingly feasible (Mir-Tutusaus et al. 2018). As one of the major components in the extracellular enzymatic machinery of white-rot fungi, MnP is capable of degrading a wide variety of contaminants and has great potential in water treatment. By encapsulating

MnP into vault nanoparticles, we observed significant improvement of BPA, BPF, and BPAP removal and remarkable decrease in reproductive toxicity of degradation products.

Unlike conventional encapsulation approaches that cause significant substrate diffusion resistance and enzyme inactivation, vault packaging had little effect on the enzymatic activity and caused negligible diffusion problems. Putting MnP into vaults led to different changes of

Km for the four tested substrates. ABTS contains two sulfonate groups, thus is easily deprotonated in solution and exhibits negative charge. In contrast, H 2O2 and guaiacol are neutral in the acidic assay buffer, and Mn 2+ is positively charged. Since vault particle exhibited a negative zeta-potential, we can infer that the electrostatic interaction between substrates and vaults resulted in the shift of K m. Negatively charged substrates like ABTS are repelled by the particle, so that their concentrations in vaults are lower than them in the solution, which leads

2+ an increase of K m. Likewise, positively charged substrate Mn is attracted and concentrated in vaults, thus it shows decreased K m for vMnP. More importantly, the similar K m between vMnP and rMnP for neutral substrates suggests vaults’ shell is permeable and mass transfer resistance of soluble substrates across the shell is negligible. Thus, for soluble BPs, which are neutral in MnP reaction buffer, vault encapsulation is unlikely to induce substrate diffusional

86 problems or K m increases. kcat values were consistent between vMnP and rMnP. Unlike the commonly used covalent binding or other strong interactions in enzyme encapsulation, which induce enzyme conformational changes and leads enzyme deactivation (Franssen et al. 2013), the antigen/antibody-like interaction affinity between the INT domain and the vaults’ shell is strong enough to keep INT-fused MnP in the vault but may not affect the folding or conformation of fused MnP enzyme, thus rMnP can maintain its activity and functionality when encapsulated inside vaults.

The removal efficiencies of four tested BPs were ranked as BPA ≈ BPF > BPAP >>

BPS, and vMnP showed higher removal rates than nMnP for all compounds except for BPS.

The absence of BPS degradation was somewhat unexpected since it has two phenolic hydroxyl groups that are favorite targets for MnP. However, similar results were also reported in a study testing natural attenuation of BPS in seawater (Danzl et al. 2009b), which showed significant biodegradation of BPA and BPF, but not of BPS. One possible explanation is that the sulfonyl group that connecting two phenol functional groups makes BPS more electronegative (Figure

S1), thus BPS tends to be electron acceptor rather than donor. It is supported by a study comparing biodegradation of various BPs under aerobic and anaerobic conditions, which showed that BPS was highly tolerant against aerobic biodegradation but was more susceptible to anaerobic biodegradation (Ike et al. 2006).

Comparing vMnP to nMnP in removing BPA, BPF, and BPAP, vMnP exhibited significantly higher transformation rates and extended functional longevity. To reduce the cost

87 of enzyme usage and provide a cost-effective enzymatic treatment approach, we employed

10-100 times lower enzyme dosage than other studies (Hirano et al. 2000b; Huang and Weber

2005), thus limited removal was observed using nMnP. It only lasted 0.5-1 hour in reactions, which was notably shorter than its storage life. The rapid enzyme inactivation can be attributed to the attack from H 2O2 (Franssen et al. 2013; Wariishi, Akileswaran, and Gold 1988), heme disruption caused by phenolic radicals (Mao et al. 2013), or heat inactivation (Wang et al.

2015b). By encapsulating in vaults, enzymatic longevity in reactions was substantially improved (at least five folds during BPA degradation), which attests that vault encapsulation not only stabilizes enzymes during storage (Wang et al. 2015b), but also protects them from

H2O2 and radical attack. The mechanism is not clear, but one possible way is to prevent heme release by hindering enzymatic conformation change upon attack (Wei et al. 2013).

The distinct production of transformation products of each BPs between vMnP and nMnP treatments is also attributed to their different stability in reactions. With enhanced functional longevity, vMnP-mediated BPs transformation accorded more closely with proposed MnP catalytic mechanism (Figure S5, S6, and S7), while the less stable nMnP resulted in different products, the majority of which were not mapped to proposed MnP-mediated pathways. It is possible that BPs transformation intermediates that were formed by MnP activities were chemically transformed by oxidizing agents in the system, such as H 2O2, which also contributed to the final product pool. For nMnP, the enzyme only survived long enough to convert BPA, BPF, and BPAP to the initial intermediates, as it was inactivated rapidly in the reactions. Afterwards, these initial intermediates were transformed mostly via chemical

88 reactions, forming the majority of the detected products, which are not in the general enzymatic transformation pathways (Figure S5, S6, and S7). For instance, in nMnP mediated

BPA transformation, the species with a m/z of 181 is probably a product from chemical oxidation of A1 (Figure S5) at the phenoxy group (Zazo et al. 2005). In contrast, vMnP lasted much longer in the reactions, which allowed the continued transformation to downstream products, such as BPA trimers or oligomers, which are relatively inert to chemical reactions and less toxic as discussed in detail below. Additionally, vMnP catalyzed reactions also consumed H 2O2, thus further reduced the chance of direct chemical oxidation.

Finally, vMnP treatment significantly reduced the toxicity of the BPs tested, unlike nMnP treatment. For this purpose, we used the nematode C. elegans because of its tractability, short reproductive period, its conservation of reproductive pathways and of reprotoxicity response to BPA (Chen, Shu, Qiu, Lee, Settle, Hee, et al. 2016; Allard and Colaiacovo 2010b).

Interestingly, we found that the ability of BPA and BPF to increase the germline apoptosis was eliminated by vMnP treatment but not by nMnP. As compared to nMnP, vMnP was more efficient in reducing the fertility impact of BPA and BPAP on nematodes. These results corroborate that vMnP not only efficiently removes the parent BPs from solution but also leads to the production of reaction intermediates and final products that are altogether less reprotoxic than the parent compounds. Divergence between vMnP and nMnP in decreasing estrogenic activity of BPs was also noted. We attribute these differences to the distinct enzymatic kinetics with differing final product profiles. The partial overlap between the reprotoxicity assays and the ER binding assay is supported by the literature on BPA which

89 shows that BPA-induced reproductive effects only partially correlate with its described weak estrogenic affinity (Maffini et al. 2006; Rubin 2011; Vandenberg et al. 2007). Altogether, these experiments therefore highlight the utility and sensitivity of the reproductive endpoints in C. elegans , which can therefore serve to assess the efficacy of various water treatment strategies.

Previous studies on advanced oxidation of BPA showed that the product toxicity is highest at earlier time points, which was attributed to the accumulation of initial oxidation intermediates (Olmez-Hanci, Arslan-Alaton, and Genc 2013; Lu et al. 2013). It is believed that these initial intermediates are more toxic, while higher-molecular weight intermediates such as BPA dimers, trimmers, or oligomers, are relatively less toxic. In this study, although very low amounts of initial intermediates in the enzymatic pathway were detected for nMnP, it is possible that these compounds were chemically converted to structurally similar compounds, which still possess reproductive toxicity. For the vMnP system, significant amount of BP oligomers were detected, suggesting the conversion of initial intermediates to less toxic polymeric products via the enzymatic pathway. Thus, our results imply that this difference in product profiles might underlie the remarkable amelioration of BPs’ toxicity following vMnP treatment.

CONCLUSIONS

This study evaluated the use of vault nanoparticles encapsulated MnP enzymes as an effective water treatment approach, and assessed the health risk of treatment products using a combination of in vivo and in vitro assays. It was demonstrated that vault encapsulation enhances enzymatic functional longevity in reactions, and lowers enzyme dosage requirement for effective removal of bisphenolic water contaminants. The reduction in BPA, BPF, and

BPAP’s reproductive toxicity in the nematode C. elegans following vMnP treatment aligns with their efficient removal after 24 hours and suggests that the process does not generate significant amounts of degradation products that also carry reproductive activity. Therefore, enzyme stabilization in vault nanoparticles combined with rigorous assessment of product toxicity opens up an exciting perspective toward the application of safe and inexpensive enzymatic systems for the treatment of bisphenols and other endocrine-disrupting compounds in water.

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the female mouse. Curr. Biol. 13 (7), 546-553. Ike, M., Chen, M.Y., Danzl, E., Sei, K., Fujita, M., 2006. Biodegradation of a variety of bisphenols under aerobic and anaerobic conditions. Water Sci. Technol. 53 (6), 153-159. Kar, U.K., Srivastava, M.K., Andersson, A., Baratelli, F., Huang, M., Kickhoefer, V.A., Dubinett, S.M., Rome, L.H., Sharma, S., 2011. Novel CCL21-vault nanocapsule intratumoral delivery inhibits lung cancer growth. PLoS One 6 (5), e18758. Kato, H., Furuhashi, T., Tanaka, M., Katsu, Y., Watanabe, H., Ohta, Y., Iguchi, T., 2006. Effects of bisphenol A given neonatally on reproductive functions of male rats. Reprod. Toxicol. 22 (1), 20-29. Kickhoefer, V.A., Garcia, Y., Mikyas, Y., Johansson, E., Zhou, J.C., Raval-Fernandes, S., Minoofar, P., Zink, J.I., Dunn, B., Stewart, P.L., Rome, L.H., 2005. Engineering of vault nanocapsules with enzymatic and fluorescent properties. Proc. Natl. Acad. Sci. U. S. A. 102 (12), 4348-4352. Lee, S., Liao, C., Song, G.J., Ra, K., Kannan, K., Moon, H.B., 2015. Emission of bisphenol analogues including bisphenol A and bisphenol F from wastewater treatment plants in Korea. Chemosphere 119, 1000-1006. Li, D.K., Zhou, Z., Miao, M., He, Y., Wang, J., Ferber, J., Herrinton, L.J., Gao, E., Yuan, W., 2011. Urine bisphenol-A (BPA) level in relation to semen quality. Fertil. Steril. 95 (2), 625-630. Liao, C.Y., Liu, F., Moon, H.B., Yamashita, N., Yun, S.H., Kannan, K., 2012. Bisphenol analogues in sediments from industrialized areas in the United States, Japan, and Korea: spatial and temporal distributions. Environ. Sci. Technol. 46 (21), 11558-11565. Lu, N., Lu, Y., Liu, F.Y., Zhao, K., Yuan, X., Zhao, Y.H., Li, Y., Qin, H.W., Zhu, J., 2013. H3PW 12 O40 /TiO 2 catalyst-induced photodegradation of bisphenol A (BPA): kinetics, toxicity and degradation pathways. Chemosphere 91 (9), 1266-1272. Maffini, M.V., Rubin, B.S., Sonnenschein, C., Soto, A.M., 2006. Endocrine disruptors and reproductive health: the case of bisphenol-A. Mol. Cell. Endocrinol. 254-255, 179-186. Mao, L., Luo, S.Q., Huang, Q.G., Lu, J.H., 2013. Horseradish peroxidase inactivation: heme destruction and influence of polyethylene glycol. Sci. Rep. 3, 3126. Meryam Sardar, R.A., 2015. Enzyme Immobilization: An Overview on Nanoparticles as Immobilization Matrix. Biochem. Anal. Biochem. 4, 1. Mir-Tutusaus, J.A., Baccar, R., Caminal, G., Sarrà, M., 2018. Can white-rot fungi be a real wastewater treatment alternative for organic micropollutants removal? A review. Water Res. 138, 137-151. Olmez-Hanci, T., Arslan-Alaton, I., Genc, B., 2013. Bisphenol A treatment by the hot persulfate process: oxidation products and acute toxicity. J. Hazard. Mater. 263, 283-290. Polka, J.K., Hays, S.G., Silver, P.A., 2016. Building Spatial Synthetic Biology with Compartments, Scaffolds, and Communities. Cold Spring Harbor Perspect. Biol. 8 (8), a024018. Rome, L.H., Kickhoefer, V.A., 2013. Development of the vault particle as a platform technology. ACS Nano 7 (2), 889-902. Rubin, B.S., 2011. Bisphenol A: an endocrine disruptor with widespread exposure and multiple effects. J. Steroid Biochem. Mol. Biol. 127 (1-2), 27-34. Stephen, A.G., Raval-Fernandes, S., Huynh, T., Torres, M., Kickhoefer, V.A., Rome, L.H.,

2001. Assembly of vault-like particles in insect cells expressing only the major vault protein. J. Biol. Chem. 276 (26), 23217-23220. Susiarjo, M., Hassold, T.J., Freeman, E., Hunt, P.A., 2007. Bisphenol A exposure in utero disrupts early oogenesis in the mouse. PLoS Genet. 3 (1), e5. Vandenberg, L.N., Hauser, R., Marcus, M., Olea, N., Welshons, W.V., 2007. Human exposure to bisphenol A (BPA). Reprod. Toxicol. 24 (2), 139-177. Wang, M., Abad, D., Kickhoefer, V.A., Rome, L.H., Mahendra, S., 2015. Vault nanoparticles packaged with enzymes as an efficient pollutant biodegradation technology. ACS Nano 9 (11), 10931-10940. Wariishi, H., Akileswaran, L., Gold, M.H., 1988. Manganese peroxidase from the basidiomycete Phanerochaete chrysosporium : spectral characterization of the oxidized states and the catalytic cycle. Biochemistry 27 (14), 5365-5370. Wei, W., Du, J.J., Li, J., Yan, M., Zhu, Q., Jin, X., Zhu, X.Y., Hu, Z.M., Tang, Y., Lu, Y.F., 2013. Construction of Robust Enzyme Nanocapsules for Effective Organophosphate Decontamination, Detoxification, and Protection. Adv. Mater. 25 (15), 2212-2218. Yamazaki, E., Yamashita, N., Taniyasu, S., Lam, J., Lam, P.K.S., Moon, H.B., Jeong, Y., Kannan, P., Achyuthan, H., Munuswamy, N., Kannan, K., 2015. Bisphenol A and other bisphenol analogues including BPS and BPF in surface water samples from Japan, China, Korea and India. Ecotoxicol. Environ. Saf. 122, 565-572. Zazo, J.A., Casas, J.A., Mohedano, A.F., Gilarranz, M.A., Rodriguez, J.J., 2005. Chemical pathway and kinetics of phenol oxidation by Fenton's reagent. Environ. Sci. Technol. 39 (23), 9295-9302.

SUPPLEMENTARY MATERIALS Characterization of Vault Encapsulated MnP Enzymes

The formation of rMnP and vault complex was confirmed by Coomassie staining and

Western blot analysis (Figure S2A). While by Coomassie staining vaults showed a clear band around 100 kD, which is the size of MVP peptides forming vaults’ shell, only rMnP was detectable by Western blot. Probably due to its large size or the effect of MnP on INT binding affinity, the copy numbers of rMnP per vault were generally lower than those of other INT fused proteins we have tested. Further examination with negative stain transmission electron microscopy revealed vMnP had identical morphology (Figure S2B) to the previously reported empty or INT bound vault nanoparticles (Kickhoefer et al. 2005). The zeta potentials between empty vaults and vaults packaged with rMnP were not significantly different (-20.50±1.29 mV for empty vaults vs. -18.65±2.45 mV for packaged vaults, Figure S2D), indicating the incorporation of rMnP did not alter vaults’ electrokinetic properties. The hydrodynamic diameters of empty and packaged vaults both centered at 50 nm (Figure S2C), but more distribution at larger diameters was observed for vaults containing rMnP, suggesting the packaging of rMnP slightly up-shifted vault particles’ hydrodynamic sizes. Additionally, the narrow distribution (45-65 nm) of both vaults also suggests that vault particles are uniformly dispersed.

Enzyme Kinetics Studies

Manganese divalent ion substrate assays were performed in 200 μL mixture containing pH 4.0 50 mM malonate buffer, MnP (5 μL of either 1.37 μM of vMnP, or 1.08 μM of rMnP

95 or 0.24 μM of nMnP), MnCl 2 (50–1000 μM), and 100μM H2O2. Assays for H 2O2 substrate were performed in the similar system but containing 1000 μM MnCl 2 and 5–100 μM H2O2.

3+ The formation of Mn -malonate was recorded by an increase in absorbance at 270 nm ( ε207nm

-1 -1 2+ = 11.59 mM cm ). Reaction rates for Mn and H 2O2 were calculated according to

3+ 2+ stoichiometry of 1mol of Mn -malonate produced per 1 mole of Mn and 0.5mol of H 2O2 consumed, respectively. ABTS kinetics assays were performed in 400 μL solution containing pH 4.0 50 mM malonate buffer, MnP (3 μL of either 1.60 μM vMnP or 0.47 μM nMnP, or 4

μL of 1.04 μM rMnP), 2 mM MnCl 2, 100 μM H2O2, and 2.5–500 μM ABTS. ABTS reaction rates were determined by measuring absorbance change rate at 420 nm, where is the

-1 -1 absorption peak of its oxidation product ( ε420nm = 36.0 mM cm ). For substrate guaiacol, the assays were carried in 50 mM malonate buffer containing MnP enzyme (12 μL of either 1.60

μM vMnP or 0.47 μM nMnP, or 16 μL of 1.04 μM rMnP), 2 mM MnCl 2, 100 μM H2O2 and guaiacol (5–125μM) with a final volume of 400 μL. Guaiacol oxidation product was

-1 -1 monitored by an increase in absorbance at 465 nm ( ε465nm = 12.1 mM cm ) to calculate guaiacol reaction rates.

Product Profile Characterizations

The analysis was performed with an ACQUITY UPLC system (Waters, Milford, MA) connected to a Waters LCT Premier mass spectrometer. UPLC separation was carried out with an ACQUITY UPLC BEH C18 column (2.1 mm x 50 mm, 1.7 μm particle size). The mobile phase was operated at 0.3 mL/min, compromising methanol and water. UPLC elution gradient started with 20% methanol (v/v) and increased to 95% (v/v) methanol at 5 minutes and

96 maintained for 1 minute. Methanol was then returned to 20% at 6.1 minutes and held for 3.9 minutes to equilibrate the column before the next injection. The mass spectrometer was operated in negative electrospray ionization (ESI) mode in the m/z 70-2000 mass range with capillary voltage 2.0 kV, desolvation gas 600 L h -1 and 350 °C, cone gas 20 L h -1 and sample cone voltage 70 V. Qualitative and quantitative analyses of UPLC/MS data were carried out using MZmine 2. Detailed data processing methods and parameters are listed in Table S1.

HPLC Analysis

The residual bisphenol concentrations were measured using a Hewlett Packard high performance liquid chromatograph (HP 1050 HPLC system) equipped with an Agilent C18 column (4.6 x 250 mm, 5 μm particle size). The mobile phase was run at 0.5 mL/min, consisting of 70% methanol, 30% H 2O and 0.1% acetic acid (v/v) for BPA and BPF, and 80% methanol, 20% H 2O and 0.1% acetic acid (v/v) for BPAP. BPA concentrations were determined by a UV detector at 277 nm, and BPF and BPAP concentrations were determined at 279 nm.

SPE Procedure

The remaining 10 mL solution was subjected to solid-phase extraction (SPE) performed using 1 cc HLB cartridges (30 mg sorbent, 30 μm particle size) from Waters (Milford, MA), which were pre-conditioned with 2 mL of methanol and 2 mL of ethanol followed by 2 mL of water. The solution was passed through the conditioned cartridge at a flow rate of 1

97 drop/second. Reaction vials were repeatedly washed (3 times) with 5 mL of water, which was also passed through the same SPE cartridges. Subsequently, cartridges were washed with 5 mL of water, followed by air dry for 10-15 minutes at about 15 bar, and then eluted with 1 mL of ethanol, which was evaporated to dryness under ultra-high purity N 2 at room temperature.

The residue was dissolved in 200 μL of ethanol, and transferred to a 300 μL glass vial and again dried under N 2 at room temperature. The final residue was reconstituted in 10 μL of ethanol, for product characterization and in vitro and in vivo toxicity tests.

SUPPLEMENTARY MATERIALS

Figure S1. Chemical Structures of BPA, BPS, BPF, and BPAP

Figure S2. Characterization of Vault Nanoparticles Packaged with MnP. (A) Vaults containing rMnP were fractionated on a 4-15% SDS-PAGE and analyzed by Coomassie staining (lane 1) and Western blotting using anti-INT antibody (lane 2). MVP and rMnP bands are indicated by arrows. (B) Negative-stained TEM image of vault particles packaged with rMnP. (C) Comparison of Zeta potentials of empty vaults and vaults containing rMnP. Error bars represent one standard error of the mean (n=10). (D) Number –based distribution of hydrodynamic diameters of empty vaults and vault particles containing rMnP.

100

2+ Figure S3. Enzyme Kinetics of vMnP, rMnP, and nMnP using Mn (A), H 2O2 (B), ABTS (C), and Guaiacol (D) as Substrates. Error bars represent one standard error of the mean (n=3).

Figure S4. No Significant BPS Degradation in 24 Hours. Error bars represent one standard error of the mean (n=3).

101

Figure S5. Proposed MnP-catalyzed BPA Transformation Pathway.

102

Figure S6. Proposed MnP-catalyzed BPF Transformation Pathway.

103

Figure S7. Proposed MnP-catalyzed BPAP Transformation Pathway.

104

Figure S8. Ethanol Vehicle Does Not Affect C. elegans Reproductive Features. Compared with the culture medium without ethanol (control), exposure to 0.1% final concentration of ethanol in medium does not significantly increase germline apoptosis (A), embryonic lethality (B), or larval lethality (C) of exposed nematodes (P>0.05). 100 µM BPA was used here as a positive control. One way-ANOVA with post-hoc Tukey HSD test performed for the comparison among all treatment groups. ***: P<0.001. N=4 for apoptosis assay and N=12-17 for embryonic lethality and larval lethality examination.

Figure S9. Larval Lethality of BPs and Their Degradation Products. The rate of larval lethality represents the percentage of larvae not surviving to the adulthood. Compared to the vehicle control (0.1% ethanol), neither exposure to untreated BP compounds nor their biodegradation products significantly increases larval lethality of nematodes. Student’s t- test performed for the comparison between the control and each 100 μM bisphenol group. One way-ANOVA with post-hoc Tukey HSD test performed for the comparison among all BPs treatment groups. N=10 -13.

105

CONTRIBUTION Preparation of vault nanoparticles encapsulated with MnP……………………….Meng Wang

Enzymatic kinetics studies…………………………………………………….….Meng Wang

Transformation of bisphenols…………………………………………………….Meng Wang

Characterization of products……………………………………………..……….Meng Wang

Estrogen receptor competitive binding assay……………………………...……Yichang Chen

Germline apoptosis assay……………………………………………………....Yichang Chen

Fertility assessment………………………………….…………………………Yichang Chen

Statistical analysis………………………………….…………Meng Wang and Yichang Chen

106

Chapter 4

BPA Antagonizes StAR-dependant Mitochondrial Cholesterol Transport to Induce Gemline

Dysfunction

107

ABSTRACT

Bisphenol A (BPA) is an endocrine disruptor used in a variety of consumer products.

Exposure to BPA has been associated with alterations in hormone synthesis and reproduction.

However, the initiating events underpinning BPA’s reproductive dysfunctions are still unclear.

Here, we address the hypothesis that BPA directly interferes with the highly evolutionary conserved process of mitochondrial cholesterol uptake, the crucial step for hormone biosynthesis. In the present study, we assessed the in vivo and biochemical relationship between BPA and mitochondrial cholesterol transporters as they relate to reproductive toxicity in the model organisms Caenorhabditis elegans (C. elegans ). Our results indicated that BPA reduced the efficiency of steroidogenic acute regulatory protein (StAR) in cholesterol transport, disturbed the intracellular cholesterol homeostasis and resulted in mitochondrial cholesterol deficiency. These changes were positively associated with the reproductive damages observed on BPA-exposed worms since restoring mitochondrial cholesterol level through exogenous cholesterol supplementation was able to rescue BPA caused damages on the reproduction of C. elegans . This study not only provides an important mechanistic piece of evidence regarding the toxicity of BPA but also highlights the importance of cholesterol in preventing BPA caused reproductive dysfunction.

108

INTRODUCTION

Bisphenol A is a widely used chemical in the manufacturing of plastics and can be found in a variety of common consumer products. Exposure to BPA is ubiquitous via food, drinks, inhalation, dental filling, and the handling of thermal paper receipts (Hartle, Navas-Acien, and

Lawrence 2016; Hines et al. 2017; Xue et al. 2018; Thayer et al. 2016). Frequent and long- term exposure to BPA renders it universally detectable in variety of human reproductive fluids and tissues such as ovarian follicular fluids and the placenta (Huang et al. 2018; Lee et al.

2018). As being one of the well-documented environmental endocrine disrupting compounds

(EDCs), exposure to BPA can impair the normal function of endocrine system and often results in reproductive dysfunction.

Exposure to BPA affects the gametogenesis and therefore impairs the reproductive outcome of an organism, which has been discovered across different species. Particularly, in rodent males, exposure to BPA leads to sperm count decrease, sperm motility impairment

(Richter et al. 2007), and sperm epigenome alteration (Doshi et al. 2012). In female rodents,

BPA in utero or neonatal exposure is also associated with inhibited germ cell nest breakdown, decreased primordial follicles (Wang, Hafner, and Flaws 2014; Zhang et al. 2012; Zhang et al.

2014), altered follicle type distribution (Gamez et al. 2015) and increased oocyte apoptosis

(Zhang et al. 2014). BPA exposure also leads to folliculogenesis defects in various species.

Increased multi-oocyte follicle number and impaired oocyte meiotic differentiation are observed in Rhesus macaques (Hunt et al. 2012) and lambs (Rivera et al. 2011). In the nematode Caenorhabditis elegans (C. elegans ), BPA exposure is associated with abnormal

109 chromosome morphogenesis, germline apoptosis, aneuploidy formation, and fertility impairment (Chen, Shu, Qiu, Lee, Settle, Que Hee, et al. 2016; Allard and Colaiacovo 2010a).

Together, these results suggest that BPA’s effects on the pathways required for germline maintenance are evolutionary conserved.

The maintenance of normal reproductive functions relies on cholesterol derivatives mediated regulation of steroid hormones in species ranging from plants to humans. BPA exposure has been shown to elicit reproductive dysfunction, it is often accompanied by hormone imbalance in an organism (Wu et al. 2013; El-Beshbishy, Aly, and El-Shafey 2013;

Xi et al. 2011; Salian, Doshi, and Vanage 2009a, 2009b; Moustafa and Ahmed 2016), but, the in-depth understanding of BPA’s impacts on hormone production and its relation to reproductive dysfunction is still insufficient.

Steroid hormone synthesis, known as steroidogenesis, follows a complex pathway by which cholesterol is converted to biologically active steroid hormones through a series of reactions mediated by the cytochrome P450 enzyme family (Miller 1988). To initiate steroidogenesis, cholesterol stored in the cytosol needs to be transported to the matrix side of the inner mitochondria membrane (IMM), where P450 cholesterol side-chain cleavage enzyme (P450scc; CYP11A1) is located (Farkash, Timberg, and Orly 1986). Cross-membrane cholesterol transport is facilitated with the help of transporter proteins localized on the outer mitochondrial membrane (OMM). These transporters include steroidogenic acute regulatory protein (StAR) and translocator protein (TSPO), both conserved across species (Fan and

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Papadopoulos 2013)

StAR is a 37-kDa protein localized on the OMM in steroidogenic cells and implicated as the rate limiting factor in mitochondrial cholesterol delivery (Ikonen 2006; Bose, Lingappa, and Miller 2002). Although the molecular mechanism of StAR-mediated cholesterol transport remains poorly understood, the indispensable role of StAR in cholesterol translocation was well demonstrated by previous studies highlighted below. For example, murine StAR knockouts displayed cytosolic cholesterol accumulation, decreased pregnenolone production, and lipoid congenital adrenal hyperplasia (CAH), a severe disorder in which afflicted individuals are unable to synthesize steroids (Caron et al. 1997).

TPSO is another cholesterol transporter highly expressed on the OMM. Early in vitro studies on mouse adrenocortical cells positioned TSPO as a critical component cooperating with StAR to deliver cholesterol into the mitochondria, based on the evidence that ligands binding with TSPO increased steroid hormone production (Krueger and Papadopoulos 1990) while inhibiting its expression reduced the steroidogenic capacity of Leydig cells (Boujrad,

Hudson, and Papadopoulos 1993). However, the role of TPSO has become controversial in recent years, as several in vivo studies have shown that normal testosterone production could still be observed in mice with a global knockout of TSPO, along with unaffected testis development, sperm count, and fertility (Tu et al. 2014; Morohaku et al. 2014). Overall, these findings indicate that TSPO is important but not necessary for the mitochondrial cholesterol transport and steroidogenesis.

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These findings indicate that mitochondrial cholesterol transport is a rate-limiting step in steroidogenesis. Its dysfunction can result in intercellular cholesterol dyshomeostasis, mitochondrial cholesterol deficiency, and hormone synthesis reduction (Hu et al. 2010).

Though previous studies showed that BPA exposure altered the expression of StAR in mouse

Leydig tumor cells (mLTC-1) (Takamiya, Lambard, and Huhtaniemi 2007) and rat granulosa cells (Samardzija et al. 2018), it was still unclear whether such alteration could affect the mitochondrial cholesterol transport, reduce hormone production and lead to reproductive dysfuntion.

C. elegans can serve as a good model organism for investigating BPA’s impact on mitochondrial cholesterol transport due to several of its unique characteristics. First, C. elegans possesses functional steroid signaling pathways that in many ways resembles those found in mammals (Wollam et al. 2012; Lopez et al. 2013). Additionally, C. elegans also relies on cholesterol derivatives as mediators to regulate its reproduction and other biological functions. Specifically, cholesterol can be metabolized by DAF-9 (homolog of CYP17A1 in human) to form dafachronic acids, that can bind and activate orphan nuclear receptor DAF-

12 (homolog of human nuclear receptors VDR/LXR) to regulate dauer diapause, reproductive development, fat metabolism, and lifespan (Mukherjee et al. 2017; Motola et al. 2006;

Matyash et al. 2004). Depletion of cholesterol in C. elegans can affect germline mitotic cell cycle and consequentially impair fertility (Merris et al. 2003). The initiation of steroidogenesis requires cholesterol mitochondrial internalization and many cholesterol transporters, such as the homologs of TSPO and StAR, were also identified in C. elegans (Fan and Papadopoulos

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2013). Last, though C. elegans relies on cholesterol to maintain its reproduction, it is unable to synthesize cholesterol de novo and thus needs to obtain cholesterol through dietary supplement (Chitwood and Lusby 1991). This feature allows us to easily manipulate the intracellular cholesterol level through controlling the amount of exogenous cholesterol in the culture medium.

In this study, we manipulated cholesterol intake in C. elegans and readily discovered an antagonistic relationship between BPA and cholesterol as administrating exogenous cholesterol restored the fertility of BPA-exposed worms. In line with this finding, we observed an imbalanced cholesterol distribution between cytosol and mitochondria in the BPA-exposed group. Additionally, by knocking out StAR, we successfully recapitulated the reproductive phenotype of BPA-exposed worms on mutants. Furthermore, BPA exposure was unable to impair the reproduction of StAR mutant, indicating StAR is crucial for BPA to elicit its toxicity.

As a conclusion, all our results indicate that BPA antagonizes StAR-dependent mitochondrial cholesterol transport to induce germline dysfunction.

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MATERIALS AND METHODS

Animal maintenance

Bristol N2 C. elegans were used as wild-type and maintained on the nematode growth medium (NGM) at 20℃. E. Coli OP50 was used as a food source. Strains with mutation on

Strl-1 F52F12.7(ok3347) I and tspo-1 C41G7.9(tm5526) I were subjected to the same assays with N2 to identify the critical mitochondrial cholesterol transporter that BPA relied on to exhibit its toxicity.

Exposure

Eggs obtained from the sodium hypochlorite treated gravid worms were placed on the

NGM plates mixed with 0, 100, and 500 µM BPA combined with 0, 0.5, and 5 µg/ml cholesterol. Exposure lasted from the birth to the end of reproductive periods of tested worms.

Ethanol was used as the vehicle and its final medium concentration was 0.1%.

Fertility assessment

To assess the fertility damage caused by the exposure, the numbers of eggs laid by each nematode during its entire reproductive period, larvae successfully hatched from eggs, and offspring surviving to adulthood were separately tallied. The percentage of unhatched eggs and the total number of living adult progeny, in terms of embryonic lethality and brood size, were calculated as they are sensitive metrics reflecting the wellness of fertility.

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Germline Apoptosis assay

After reaching adulthood, worms were incubated in M9 solution with 25 µg/ml of acridine orange (AO) for 2 hours at room temperature (Allard and Colaiacovo 2010a). AO specifically labels engulfed apoptotic germline nuclei and emits 525 nm fluorescence after excitation. The total number of apoptotic nuclei in the pachytene zone of the posterior gonad of each worm was counted. Apoptotic index was calculated as the percentage of apoptotic nuclei divided by the total number of nuclei in the pachytene zone.

Germline morphology examination

To examine the exposures caused germline morphological alterations, whole mount nuclei staining was performed on exposed worms. Briefly, young adult worms were firstly fixed by the Carnoy’s fixation and then subjected to DAPI staining. Images from the posterior gonad of each worm were captured and analyzed. The numbers of germline nuclei in each meiotic stage were tallied separately.

Mitochondria Isolation and purification

Mitochondria of nematodes were extracted with the use of Mitochondria Isolation Kit

(Sigma-Aldrich, Cat. MITOISO1). In general, 2 g of worms from each exposure group was collected, rinsed with M9 solution, incubated with lysis buffer EBA containing trypsin for 20 mins and then homogenized on ice. Lysates were centrifuged at 11000 g for 10min at 4℃. The precipitates were re-suspended and collected as the crude mitochondria extraction with lysosomes. To purify the collected crude mitochondria, sucrose density gradient

115 ultracentrifugation was performed as described before (Clayton and Shadel 2014). Purified mitochondria were collected, re-suspended with PBS buffer and then stored at -80℃ till use.

Cholesterol Assay

The concentration of cholesterol in mitochondria of worms from each exposure group was measured with the use of Amplex TM Red Cholesterol Assay kit (Invitrogen TM , Cat.

A12216). Briefly, cholesterol esters in the mitochondria was firstly hydrolyzed by cholesterol esterase into cholesterol, which was then oxidized by cholesterol oxidase to yield H 2O2. The

R○ H2O2 then reacted with Amplex Red reagent in the presence of horseradish peroxidase and yielded highly fluorescent resorufin with emission maxima of approximately 595 nm. The cholesterol level of a sample could be measured based on the fluorescent intensity of resorufin.

The protein level of each sample was measured as well with the use of Bio-Rad DC TM Protein

Assay Kit to correct the cholesterol level.

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RESULTS

Exogenous cholesterol rescues BPA induced fertility damage

Cholesterol deficiency impairs worms’ fertility (Merris et al. 2003). If BPA impairs fertility by causing a cholesterol deficiency, then exogenous cholesterol administration should alleviate such damage. To test this hypothesis, the effect of BPA and cholesterol on the fertility of wild-type N2 worms were first assessed. The total number of eggs produced, the percentage of unhatched eggs (embryonic lethality), and the number of progeny successfully reaching adulthood (brood size) per worm were examined since they are sensitive fertility metrics

(Allard and Colaiacovo 2010a). In absence of cholesterol, we observed a concentration- dependent decrease in egg number and brood size, and increase in embryonic lethality, the latter two reaching significance (P<0.001) at the highest concentration of 500 μM of BPA when compared to the control (Fig.1). By contrast, in the presence of cholesterol (0.5 μg/ml or 5 μg/ml), no significant difference was observed between the control and the two BPA concentrations for all three fertility measures (Fig. 1). These results indicate that BPA induced reproductive impact is cholesterol level dependent.

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Figure 1. Exogenous cholesterol rescues BPA induced fertility damage. (A) The total number of eggs laid by each individual worm. (B) Embryonic lethality as the percent of unhatched eggs. (C) Brood size as the number of offspring reaching adulthood. N=8-19 for each group. One-way ANOVA with Turkey Post hoc test. *** P<0.001.

BPA-induced germline dysfunction is cholesterol level dependent.

A well-conserved feature of BPA’s impact on reproduction is its alteration of critical meiotic events such as synaptonemal complex formation and meiotic recombination, which result in increased germline apoptosis and nuclear loss (Allard and Colaiacovo 2010a). We therefore assessed whether exogenous cholesterol would also rescue these hallmarks of BPA exposure on the germline. Compared to the control group, exposure to 500 μM of BPA alone significantly reduced the total germline nuclei number in N2 worm by 16% (P<0.05), resulting in germline shrinkage and germline morphological abnormalities (Fig. 2A). A cluster of germline nuclei loss forming a gap in the field of nuclei was more frequently observed in BPA- exposed worms compared to control worms (Fig. 2B). 500 μM of BPA significantly increased

118 the frequency of gaps by 2.8-fold (P<0.001) (Fig. 2C). These findings were in accordance with the apoptosis assay result as 500 μM of BPA increased germline apoptosis by 200%

(P<0.001) in comparison to the control (Fig. 2D and E). On the contrary, exposure to 5 μg/ml of cholesterol significantly increase the total germline nuclei number by 130% (P<0.05). By contrast, addition of 5 μg/ml cholesterol dramatically rescued BPA’s induction of germline gaps and apoptosis (Fig. 2). In summary, these findings, coupled with the fertility assessment results, suggest that BPA exposure caused reproductive damage is cholesterol related and can be antagonized by exogenous cholesterol supplementation.

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Figure 2. Cholesterol prevents BPA induced germline damage. (A) The total number of germline nuclei in the posterior gonad. N=5 worms per repeat, 4-9 repeats per treatment group. (B) Low magnification images of DAPI-stained gonads from age-matched hermaphrodites exposed to vehicle control (0.1% ethanol), 500 μM BPA, 5 μg/ml cholesterol or a combination of these two. Red arrowhead indicates a gap with germline nuclear loss (Scale bar, 10 μm). (C) Quantification of germline nuclear loss frequency in the gonad of worms exposed to vehicle or to the indicated doses of BPA and/or cholesterol. N = 20 worms per repeat, 4-8 repeats per treatment group. (D) Apoptotic index, i.e. the number of apoptotic nuclei number per 100 pachytene nuclei in the gonad of examined worms. N = 20 worms per repeat, 4-9 repeats per treatment. (E) Low-magnification images of acridine orange-stained gonads from age-matched hermaphrodites exposed to vehicle control (0.1% ethanol), 500 μM BPA, 5 μg/ml cholesterol or a combination of these two. (Scale bar, 100 μm). One-way ANOVA with Turkey Post hoc test. * P<0.05 and *** P<0.001.

BPA disrupts intracellular cholesterol homeostasis

Though exogenous cholesterol administration restores the fertility of worms exposed to

BPA, the role of mitochondria in this process is remains unclear. C. elegans , like other species, relies on cholesterol derivatives dafachronic acid to regulate reproduction (Wollam et al. 2012).

Since the initial step of cholesterol conversion happens in mitochondria (Dumas et al. 2010),

BPA may reduce the amount of available cholesterol to mitochondria, affect cholesterol conversion and hence impair worms’ reproduction. To test this hypothesis, the effect of BPA on intracellular cholesterol distribution was investigated by measuring the total intracellular and mitochondrial cholesterol levels. As shown in Figure 3, the total intracellular cholesterol level was not affected by exogenous cholesterol administration and it consistently remained at 19 μg/mg, normalized to protein level. Compared with the control, exposure to 500 μM

BPA significantly increased the total intercellular cholesterol level by 1.5-fold (P<0.05) regardless of the cholesterol level in the media (Fig. 3A). By contrast, we observed a concentration-dependent decrease in mitochondrial cholesterol level in all groups exposed to

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BPA. This change was most significant in worms exposed to 500 μM BPA without additional cholesterol exposure in the comparison with the control (Fig. 3B). Overall, these results indicate that BPA disrupts intracellular cholesterol homeostasis and imply the ability of BPA to interfere with the mitochondrial cholesterol uptake.

Figure 3. BPA disrupts intracellular cholesterol homeostasis. (A) Total intracellular cholesterol level of worms exposed to vehicle control (0.1% ethanol) or indicated concentrations of BPA and/or cholesterol. (B) Cholesterol level measured from the extracted mitochondria of worms exposed to the indicated concentrations of BPA and cholesterol. Cholesterol level was normalized by the protein concentration of each sample. N=7-12 for each group. One-way ANOVA with Turkey Post hoc test. * P<0.05 and ** P<0.01.

BPA relies on StAR but not TSPO to elicit its reproductive toxicity

StAR and TSPO are the major cholesterol transporters on the mitochondrial outer membrane, delivering cholesterol to the mitochondrial inner matrix to initiate the biosynthesis.

If BPA impairs the delivery efficiency of these two transporters and therefore decreases the mitochondrial cholesterol level, then knocking out the targeted transporter in a worm will resemble the reproductive dysfunction caused by BPA exposure on this individual.

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Furthermore, since the BPA affected transporter does not exist in the knockout mutant, thus

BPA won’t induce any further reproductive damage on the mutant to exacerbate its already compromised fertility. Hence, nematodes with mutation on StAR and TSPO were subjected to fertility assessment and germline morphology examination following the same exposure conditions described above.

As the result shown in figure 4, the overall reproductive ability of StAR mutants was reduced when compared to that of wild type nematodes, evidenced by increased embryonic lethality, reduced brood size, decreased germline nuclei number, and elevated germline apoptosis (P<0.05) similar to those of BPA. In conclusion, knocking out StAR resembles the adverse effects of BPA on the reproduction of N2 worms. On the other hand, exposure to BPA didn’t worsen the fertility of StAR mutants as there was no significant difference observed between exposed and unexposed groups in egg number, embryonic lethality, or brood size

(Fig. 4A to C). Similarly, no adverse effect of BPA was found on StAR mutants in the germline morphology, germline size, frequency of gaps, and germline apoptosis of exposed worms are close to those in the control group (Fig. 4D to F). These findings indicate that StAR is a crucial mediator for BPA to elicit its reproductive toxicity.

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Figure 4. StAR is a crucial mediator for BPA to elicit its reproductive toxicity. (A) The total number of eggs laid by each individual worm. N=10-11 for each group. (B) Embryonic lethality is the percent of unhatched eggs. N=10-11 for each group. (C) Brood size is the number of offspring reaching adulthood. N=10-11 for each group. (D) The total number of germline nuclei in the posterior gonad of examined worms. N=5 worms per trail, five to seven repeats per treatment group. (E) Quantification of germline nuclear loss frequency in the gonad of worms exposed to vehicle or to the indicated doses of BPA and/or cholesterol. N=20 worms per trial, five to seven repeats per treatment group. (F) Apoptotic index of worms in each treatment. N=20 worms per trial, five to seven repeats per treatment. One- way ANOVA with Turkey Post hoc test. * P<0.05 and *** P<0.001. Student’s t-test between N2 and StAR mutant. * P<0.05.

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On the contrary, knocking out TSPO failed to resemble the reproductive damage caused by BPA exposure on the wild-type. In addition, among TSPO mutants, compared to the unexposed control group, 500 μM of BPA significantly increased the embryonic lethality and reduced the brood size (P<0.05) (Fig. 5A to C). In line with these findings, the total germline nuclei number was also significantly reduced, accompanied with increased germline apoptosis and gaps frequency (Fig. 5D to E). Additionally, like what had been observed on N2 worms, exogenous cholesterol antagonized the reproductive toxicity of BPA on TSPO mutants and brought all the examined metrics down to same levels of the control group (Fig. 5). Hence, these results indicate that TSPO is not necessary for BPA to elicit its reproductive toxicity.

Together, our results indicate that BPA relies on interacting with StAR but not TPSO to exhibit its reproductive toxicity. It also implies that BPA may work through affecting the cholesterol delivery efficiency of StAR to disturb the intracellular cholesterol homeostasis and therefore impair the reproductive function of an organism.

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Figure 5. TSPO is not necessary for BPA to elicit its reproductive toxicity. (A) The total number of eggs laid by each individual worm. N=10-11 for each group. (B) Embryonic lethality is the percent of unhatched eggs. N=10-11 for each group. (C) Brood size is the number of offspring reaching adulthood. N=10-11 for each group. (D) The total number of germline nuclei in the posterior gonad of examined worms. N=5 worms per trail, five to seven repeats per treatment group. (E) Quantification of germline nuclear loss frequency in the gonad of worms exposed to vehicle or to the indicated doses of BPA and/or cholesterol. N=20 worms per trial, five to seven repeats per treatment group. (F) Apoptotic index of worms in each treatment. N=20 worms per trial, five to seven repeats per treatment. One- way ANOVA with Turkey Post hoc test. * P<0.05, ** P<0.01, and *** P<0.001. Student’s t-test between N2 and TSPO mutant. * P<0.05.

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DISCUSSION

Cholesterol derivatives act as crucial mediators to regulate vital biological functions of an organism, such as development and reproduction. Although BPA is a well-documented endocrine disruptor and possesses reproductive toxicity, its impact on the early event of steroidogenesis, mitochondrial cholesterol internalization, is still unclear. In this study, we first demonstrated an antagonistic relationship between BPA and cholesterol as exogenous cholesterol supplementation rescued all BPA-mediated reproductive dysfunctions in C. elegans. We further assessed the intracellular cholesterol level and found a positive association between intracellular cholesterol dyshomeostasis and BPA-elicited reproductive damage. In the end, with the use of different knockout strains, we identified mitochondrial cholesterol transporter StAR as the key mediator required by BPA to exhibit its reproductive toxicity. It is worth noting that the doses of BPA used in this study were based on our previous studies

(Allard and Colaiacovo 2010a; Chen, Shu, Qiu, Lee, Settle, Que Hee, et al. 2016) in order to generate a range of internal doses in C. elegans which would approximate organ BPA levels in human follicular and fetal tissue such as placental (Schonfelder et al. 2002) and umbilical cord samples (Vandenberg et al. 2013).

Though being evolutionarily distant to human and other species, C. elegans still relies on cholesterol derivatives to regulate many biological functions, such as development, reproduction and aging (Mukherjee et al. 2017; Motola et al. 2006; Matyash et al. 2004).

Deprivation of cholesterol impairs the fertility of worms, causes germline dysfunction and results in sterility (Merris et al. 2003). Since C. elegans is unable to de novo synthesize

126 cholesterol, exogenous cholesterol through dietary supplementation is the only route for worms to obtain cholesterol (Motola et al. 2006). BPA exposure affects critical meiotic events, such as synaptonemal complex formation and meiotic recombination related double-strand break repair, triggers checkpoint activation and results in germline dysfunction and fertility damage (Allard and Colaiacovo 2010a; Chen, Shu, Qiu, Lee, Settle, Que Hee, et al. 2016;

Susiarjo et al. 2007b; Hunt et al. 2012). This study is the first one to report that exogenous cholesterol can rescue BPA exposure induced germline dysfunction and restore the fertility of exposed organisms. A similar antagonistic relationship between BPA and cholesterol was observed on rodent granulosa cells before as administrating cholesterol prevented BPA’s cytotoxicity and restored the ability of exposed cells to produce of progesterone, the precursor of steroid hormone (Samardzija et al. 2018). Our results, combined with the previous study, suggest a possible toxic mechanism that BPA impairs the reproductive function of an organism through reducing the available cholesterol to its conversion, steroidogenesis.

The impact of BPA on sterioidogenesis has been well studied before. Exposure to BPA alters the expression of critical enzymes in hormone biosynthesis (Kwintkiewicz et al. 2010;

Mansur et al. 2016; Samardzija et al. 2018) and impairs the synthesis of steroid hormone and its precursor in both animal (Peretz et al. 2011; Samardzija et al. 2018) and human granulosa cells (Kwintkiewicz et al. 2010). However, whether mitochondrial cholesterol uptake, the initial step in steroidogenesis, can be affected by BPA has not been comprehensively studied.

By measuring the intracellular and mitochondrial cholesterol level of worms exposed to varying concentrations of BPA and/or cholesterol, we observed a cytosolic cholesterol

127 accumulation caused by BPA exposure, evidenced by increased total intracellular cholesterol level and decreased mitochondrial cholesterol level. This change is consistent with previous finding that exposure to BPA results in cholesterol sequestration to the perinuclear area in rodent granulosa cells (Samardzija et al. 2018). Although accumulating evidence suggests a role of BPA in lipid metabolic disorders and shows BPA exposure leads to hepatic lipid accumulation (Marmugi et al. 2012; Ke et al. 2016) and hypercholesterolemia (Marmugi et al.

2014), its in-depth mechanism is still unclear. Though the increased cytosolic cholesterol level could be explained by BPA’s ability to decrease the expression of ATP-binding cassette transporters ABCA1, which is responsible for the efflux of cholesterol (Samardzija et al. 2018).

However, it cannot explain the decreased mitochondrial cholesterol level since ABCA1 deficiency allows for increased mitochondrial cholesterol (Smith and Land 2012). Therefore, we speculated that the mitochondrial cholesterol transporter was affected by BPA.

To initiate steroidogenesis, cholesterol needs to be transported to the inner mitochondrial membrane where cholesterol side-chain cleavage enzyme (CYP11A1, P450scc) and 3β- hydroxysteroid dehydrogenase (CYP17A1, 3β-HSD) reside (Farkash, Timberg, and Orly

1986). Homologs of these enzymes serving same purpose have been identified in C. elegans

(Dumas et al. 2010; Fan and Papadopoulos 2013), suggesting that cholesterol also needs to be transported in mitochondria for worms to synthesize its unique sterol hormones, known as dafachronic acids (Wollam et al. 2012).

Cholesterol mitochondrial transport requires the facilitation of StAR and TSPO (Stocco

128 et al. 2017). Before our study, it has been observed that BPA exposure increases the expression of StAR (Takamiya, Lambard, and Huhtaniemi 2007; Samardzija et al. 2018). This could be due to the down-regulation effect of BPA on the expression of microRNA Let-7 family (Veiga-

Lopez et al. 2013), which repressively controls the expression of StAR (Men et al. 2017).

However, the increased expression of StAR conflicts with accompanied progesterone decrease in the same organism (Samardzija et al. 2018; Zhou et al. 2008). Combined with the observed cytosolic cholesterol accumulation (Samardzija et al. 2018), all suggest that BPA impairs the efficiency of StAR in cholesterol transport. In line with this notion, our results showed that knocking out StAR resembled the reproductive dysfunction caused by BPA exposure on the wild type worms. Furthermore, BPA was unable to induce any further reproductive damage on the StAR mutant to worsen its already compromised fertility, proving StAR is a crucial mediator for BPA to elicit its reproductive toxicity. Therefore, the increased expression of

StAR observed in previous studies could be a consequence of the positive feedback in mitochondria to mitigate the stress from cholesterol deprivation. On the contrary, knocking out TSPO failed to resemble BPA exposure caused reproductive damage and exposure to BPA was still able to impair the reproduction of TSPO mutant. Together, all these results indicate that BPA relies on StAR but not TSPO to elicit its reproductive toxicity

As a summary, our study here demonstrates that BPA exposure is able to affect the efficiency of StAR in mitochondrial cholesterol transport, alters intracellular cholesterol distribution and thus impairs the reproduction of an organism. This study not only provides an important mechanism piece of evidence regarding the effect of BPA on steroidogenesis and

129 reproduction but also highlights the importance of cholesterol in preventing BPA caused reproductive dysfunction. Individual with lipid metabolism disorder, mutation in StAR, or low cholesterol level could become sensitive to BPA’s reproductive toxicity.

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CONTRIBUTION Fertility assessment………………………………………Yichang Chen and Katherine Gibbs

Germline apoptosis assay…………………………………………………….....Yichang Chen

Germline morphological examination….Yichang Chen, Katherine Gibbs, and Aleena Hussain

Mitochondria extraction…Yichang Chen, Katherine Gibbs, Blake Panter, and Aleena Hussain

Cholesterol measurement…..………Yichang Chen, Katherine Gibbs, and Robert Damoiseaux

Statistical analysis…………………………….…….…………………………..Yichang Chen

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Chapter 5

Summary and Discussion

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Bisphenol A (BPA) is a chemical that has been widely used in the production of plastics for more than half a century and it presents in many parts of our daily lives. People can be ubiquitously exposed to BPA via food (Liao and Kannan 2014), drinks (Jin and Zhu 2016), dental restoration (Fleisch et al. 2010), or even from receiving receipts from grocery stores

(Thayer et al. 2016). The concerns about BPA’s safety has never disappeared from the public eye since 1997 when the low-dose adverse effect of BPA was first reported (vom Saal et al.

1997).

The intense scrutiny from the public regarding the potential health risks of BPA led to its substitution with structural analogs to make “BPA-Free” products. However, most of the BPA substitutes still possess weak estrogenic activity (Hashimoto and Nakamura 2000) and their toxicities, especially at a whole-organism level, are still unknown. Investigating the reproductive toxicity of BPA-substitutes with the use of traditional animal models is both labor-intensive and time-consuming. A standardized reproductive toxicity assessment coasts at least three months and a hundred rodents to identify the single-generation health risk of one chemical (EPA 1991). By contrast, C. elegans , a short-lived nematode, has many advantages over the traditional animal models, such as low cost, ease of maintenance, large brood size, and conserved biological processes and signaling pathways with other species (Leung et al.

2008). All these make C. elegans a suitable alternative model organism for assessing a chemical’s reproductive toxicity risk.

Therefore, in the first part of this study, we investigated and compared the reproductive toxicity of BPA and Bisphenol S (BPS) with the use of C. elegans . BPS is one of the major

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BPA-substitutes used in the manufacture of receipts and other products (Liao, Liu, and Kannan

2012; Liao and Kannan 2014). It can be consistently detected from human bio-fluid samples and in the environment (Liao, Liu, Moon, et al. 2012; Liao, Liu, Alomirah, et al. 2012). In the

United States, the level of BPS found in human urine samples gradually increased with year

(Ye et al. 2015). In Japan, the human estimated daily intake of BPS has already surpassed that of BPA (Liao, Liu, Alomirah, et al. 2012). The increased usage of BPS around the world makes it crucial for us to develop a better understanding of its toxic effects. Meanwhile, the combined effect of BPA and BPS was also studied because we are exposed to both of them at the same time now (Liao and Kannan 2014; Liao, Liu, Guo, et al. 2012; Liao, Liu, Moon, et al. 2012).

In our study, we found that similar to BPA, BPS causes severe reproductive defects including germline apoptosis and embryonic lethality. However, the results from meiotic recombination, targeted gene expression, whole transcriptome, ontology analyses as well as biological similarity analysis based on the EPA’s ToxCast database all indicate that the deleterious effects of BPS are partially achieved via mechanism distinct from BPA’s.

Meanwhile, most of the results indicate that the combined effect of BPA and BPS is additive, suggesting the major signaling pathways affected by BPA and BPS are shared. In addition, the estrogenic activity of bisphenols is often considered as the main cause for them to disrupt the endocrine system of an organism and further induce reproductive dysfunction (Le Fol et al.

2017). Though a few of nuclear hormone receptors in C. elegans have been found sharing sequence similarity with the human estrogen receptor, none of them is able to fully interact with or response to estradiol (Mimoto et al. 2007; Fischer et al. 2012). Therefore, this unique

138 feature of C. elegans , combined with the observed toxic effect of BPA and BPS in this study, suggests that the reproductive toxicity of bisphenols could not completely stem from their estrogenic activity. Furthermore, the impact of BPA on reproduction is trans-generational and can be passed down to subsequent generations through heritable epigenetic alterations

(Camacho et al. 2018). Whether BPS and other BPA-substitutes also possess a similar trans- generational effect and what the combined effect of bisphenols would be in the indirectly exposed subsequent generations are still unknown and worth investigating in the future. As a conclusion, our findings here raise new concerns about the safety of using BPA alternatives and the risks associated with human exposure to their mixtures.

The first part of this study highlights the value of C. elegans as a relevant model, useful model for evaluating the toxicity of BPA and its substitutes. Therefore, in the second part of this study, we utilized C. elegans to evaluate the detoxification efficiency and safety of a novel enzymatic bisphenol biodegradation system developed by Dr. Shaily Mahendra and her collaborators at UCLA (Wang et al. 2015). Manganese peroxidase (MnP) was utilized in this system to degrade bisphenol compounds (BPs) found in water. While the vault nanoparticles packaged MnP (vMnP) showed a better performance than its native unpacked form (nMnP) in degrading BPA, BPF, and BPAP, the nanoparticles encapsulation also altered the reaction kinetics and resulted in a less toxic product profile than its unpacked form, as supported by the results from both in vivo and in vitro toxicity assessments. Here, C. elegans was applied as the model organism in the in vivo assessment. Our result showed that treatment mediated by vMnP performed better than nMnP in removing the reproductive toxicity of BPs. In

139 contrast, the result of in vitro assay indicated that nMnP possessed a better ability than vMnP in removing the estrogenic activity of BPs. Though the reproductive toxicity of bisphenol does not completely stem from its estrogenic activity, this finding emphasizes the necessary of employing another model organism to obtain a more comprehensive evaluation on the detoxification efficiency and safety of the vault nanoparticle packaged manganese peroxidase.

On the other hand, though our results indicate that vMnP is overall better than nMnP in both degrading BPs and removing their toxicity, one drawback limiting the application of this system in water treatment is the inability of MnP in degrading BPS. This can be caused by the distinct chemical structure of BPS from those of other BPs as the two phenolic rings are bridged by a sulfonyl group but not an alkyl group, which is the target of MnP (Hirano et al.

2000). The resistance of BPS towards degradation was also observed in other water treatment methods, such as constructed wetlands system (Wirasnita, Mori, and Toyama 2018) and microbial biodegradation system (Sakai et al. 2007; Danzl et al. 2009). So far, only

Sphingobium fuliginis (S. fuliginis ) OMI mediated treatment has shown the ability to decompose BPS in sludge (Ogata et al. 2013). To isolate the effective enzymes from S. fuliginis OMI and encapsulate them with vault nanoparticles could be the approach applied in the future to remediate BPS in water. Examining the safety, detoxification efficiency and compatibility with vMnP of those enzymes is necessary and would become one part of our future study.

140 assessment for evaluating the toxicities of BPA’s substitutes. As being a well-documented endocrine disruptor, BPA can interfere with the endocrine system and affect the reproduction of an organism (Viguie et al. 2018). Though such effects have been observed in many model organisms under experimental conditions, the toxic mechanism of BPA on hormone synthesis, specifically its initial step during mitochondrial cholesterol uptake, is still unclear. Therefore, in the last part of our study, we assessed the in vivo and biochemical relationship between BPA and mitochondrial cholesterol transporters as they relate to reproductive toxicity in C. elegans .

Our results indicated that BPA reduced the efficiency of the steroidogenic acute regulatory protein (StAR) in cholesterol transport, disturbed the intracellular cholesterol homeostasis and resulted in mitochondrial cholesterol deficiency. These changes were positively associated with the reproductive damages observed on BPA-exposed worms since restoring mitochondrial cholesterol level through exogenous cholesterol supplementation was able to rescue the reproductive dysfunctions in BPA-exposed worms. However, whether BPA impairs the cholesterol mitochondrial internalization through competing with cholesterol in utilizing

StAR or directly affecting the availability of StAR to cholesterol transport is still unknown.

Measuring the BPA level in mitochondria and the expression level of StAR would be approaches applied in the future to answer this question. A decreased mitochondrial cholesterol level accompanied with an increased BPA level in mitochondria without any alteration in the expression of StAR would clearly indicate a competitive relationship between

BPA and cholesterol on the utilization of StAR. Overall, our findings here not only provide an important mechanistic piece of evidence regarding the toxicity of BPA, but also highlights the importance of cholesterol in preventing BPA caused reproductive dysfunction. Individual with

141 low cholesterol level or StAR mutation would be more sensitive to BPA’s toxicity.

In conclusion, the work presented here not only expands our knowledge on the reproductive toxicity of bisphenol compounds but also clearly highlights the need for more comprehensive testing on substitutes-selected from structural analogues in functional in vivo assays such as those described here with C. elegans . In addition, this work also highlights the value of C. elegans as a good in vivo assay model in the study of reproductive toxicology, especially in applications, such as evaluating the detoxification efficiency and safety of bisphenols treatment methods.

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