Reproductive toxicity of emerging plasticizers, flame retardants, and bisphenols, using culture of the rat fetal testis†.

The use of bis (2-ethylhexyl) phthalate (DEHP), 2,2'4,4'-tetrabromodiphenyl ether (BDE47), and bisphenol A (BPA), as plasticizers, flame retardants, and epoxy resins, respectively, has been regulated due to their endocrine disrupting activities. Replacements for these chemicals are found in human matrices, yet the endocrine disrupting potential of these emerging contaminants is poorly characterized. We compared the effects of legacy chemicals with those of their replacements using fetal rat testis organ culture. Fetal testes sampled at gestation day 15 were grown ex vivo, and the impact was evaluated after a 3-day exposure to 10 µM of each legacy chemical; two BPA analogs (bisphenol M and bisphenol TMC); three replacements for DEHP/MEHP (2,2,4-trimethyl-1,3-pentanediol diisobutyrate, diisononyl-phthalate, and diisodecyl adipate); or two replacements for BDE47 (tributoxyethyl phosphate and isopropylated triphenyl phosphate). We showed that only BPA and MEHP significantly decrease testosterone secretions after 24 h, while BPM and BPTMC have the opposite effect. Luteinizing hormone-stimulated testosterone was reduced by BPA and MEHP but was increased by BPTMC. After exposure, testes were used for immunofluorescent staining of germ cells, Sertoli cells, and Leydig cells. Interestingly, exposures to BPM or BPTMC induced a significant increase in the Leydig cell density and surface area. A decrease in germ cell density was observed only after treatment with MEHP or BDE47. MEHP also significantly decreased Sertoli cell proliferation. These studies show that some replacement chemicals can affect testicular function, while others appear to show little toxicity in this model. These findings provide essential information regarding the need for their regulation.

High-content imaging analyses of the effects of bisphenols and organophosphate esters on TM4 mouse Sertoli cells†.

The endocrine disruptive effects of bisphenol A (BPA) and brominated flame retardants (BDE-47) have led to restrictions on their use and increased the pressure to identify safe replacements for these chemicals. Although there is evidence that some of these alternatives may be toxic to spermatogonial and Leydig cells, little is known about the toxicity of emerging replacements on Sertoli cells. We used high-content imaging to compare the effects of legacy chemicals, BPA and BDE-47, to their corresponding replacements. TM4 Sertoli cells were exposed for 48 h to each chemical (0.001-100 µM) followed by cytotoxicity and phenotypic endpoint assessment. The benchmark concentration potency ranking for bisphenols based on cytotoxicity was BPTMC > bisphenol M > BPAF>BPF > BPS > BPA. Human administered equivalent dose (AED) determination ranked BPS as the most potent alternative replacement. The benchmark concentration potency ranking of BDE-47 and organophosphate esters based on cytotoxicity was TDtBPP>BDMPP>TBOEP>TDCPP>TMPP>TPHP>BDE47>IPPP=BPDP=TCPP. Additionally, TM4 cell exposure to BDE-47 increased Calcein intensity (57.9 µM) and affected lysosomes (21.6 µM), while exposure to TPHP and TMPP resulted in cellular oxidative stress changes at benchmark concentration values as low as 0.01 and 0.4 µM, respectively. Overall bioactivity considerations of the chemicals on TM4 via ToxPi analyses and AED modeling further validated emerging replacements as highly potent chemicals in comparison to BPA and BDE-47. These findings demonstrate that many bisphenol and flame retardant replacements are more potent in Sertoli cells than the legacy chemical they are replacing and that phenotypic parameter assessment is an effective tool in chemical toxicity assessment.

Phthalates and alternative plasticizers differentially affect phenotypic parameters in gonadal somatic and germ cell lines†.

The developmental and reproductive toxicity associated with exposure to phthalates has motivated a search for alternatives. However, there is limited knowledge regarding the adverse effects of some of these chemicals. We used high-content imaging to compare the effects of mono (2-ethylhexyl) phthalate (MEHP) with six alternative plasticizers: di-2-ethylhexyl terephthalate (DEHTP); diisononyl-phthalate (DINP); di-isononylcyclohexane-1,2-dicarboxylate (DINCH); 2-ethylhexyl adipate (DEHA); 2,2,4-trimethyl 1,3-pentanediol diisobutyrate (TXIB) and di-iso-decyl-adipate (DIDA). A male germ spermatogonial cell line (C18-4), a Sertoli cell line (TM4) and two steroidogenic cell lines (MA-10 Leydig and KGN granulosa) were exposed for 48 h to each chemical (0.001-100 µM). Cell images were analyzed to assess cytotoxicity and effects on phenotypic endpoints. Only MEHP (100 µM) was cytotoxic and only in C18-4 cells. However, several plasticizers had distinct phenotypic effects in all four cell lines. DINP increased Calcein intensity in C18-4 cells, whereas DIDA induced oxidative stress. In TM4 cells, MEHP, and DINCH affected lipid droplet numbers, while DEHTP and DINCH increased oxidative stress. In MA-10 cells, MEHP increased lipid droplet areas and oxidative stress; DINP decreased the number of lysosomes, while DINP, DEHA, and DIDA altered mitochondrial activity. In KGN cells, MEHP, DINP and DINCH increased the number of lipid droplets, whereas DINP decreased the number of lysosomes, increased oxidative stress and affected mitochondria. The Toxicological Priority Index (ToxPi) provided a visual illustration of the cell line specificity of the effects on phenotypic parameters. The lowest administered equivalent doses were observed for MEHP. We propose that this approach may assist in screening alternative plasticizers.

A cross-species comparative approach to assessing multi- and transgenerational effects of endocrine disrupting chemicals.

A wide range of chemicals have been identified as endocrine disrupting chemicals (EDCs) in vertebrate species. Most studies of EDCs have focused on exposure of both male and female adults to these chemicals; however, there is clear evidence that EDCs have dramatic effects when mature or developing gametes are exposed, and consequently are associated with in multigenerational and transgenerational effects. Several publications have reviewed such actions of EDCs in subgroups of species, e.g., fish or rodents. In this review, we take a holistic approach synthesizing knowledge of the effects of EDCs across vertebrate species, including fish, anurans, birds, and mammals, and discuss the potential mechanism(s) mediating such multi- and transgenerational effects. We also propose a series of recommendations aimed at moving the field forward in a structured and coherent manner.

Androgens Upregulate Pathogen-Induced Placental Innate Immune Response.

Group B Streptococcus (GBS) is a leading cause of placental infection, termed chorioamnionitis. Chorioamnionitis is associated with an increased risk of neurobehavioral impairments, such as autism spectrum disorders, which are more prominent in males than in female offspring. In a pre-clinical model of chorioamnionitis, a greater inflammatory response was observed in placenta associated with male rather than female fetuses, correlating with the severity of subsequent neurobehavioral impairments. The reason for this sex difference is not understood. Our hypothesis is that androgens upregulate the placental innate immune response in male fetuses. Lewis dams were injected daily from gestational day (G) 18 to 21 with corn oil (vehicle) or an androgen receptor antagonist (flutamide). On G 19, dams were injected with saline (control) or GBS. Maternal, fetal sera and placentas were collected for protein assays and in situ analyses. Our results showed that while flutamide alone had no effect, a decrease in placental concentration of pro-inflammatory cytokines and infiltration of polymorphonuclear cells was observed in flutamide/infected compared to vehicle/infected groups. These results show that androgens upregulate the placental innate immune response and thus may contribute to the skewed sex ratio towards males observed in several developmental impairments resulting from perinatal infection/inflammation.

Aging and oxidative stress alter DNA repair mechanisms in male germ cells of superoxide dismutase-1 null mice.

The efficiency of antioxidant defense system decreases with aging, thus resulting in high levels of reactive oxygen species and DNA damage in spermatozoa. This damage can lead to genetic disorders in the offspring. There are limited studies investigating the effects of the total loss of antioxidants, such as superoxide dismutase-1 (SOD1), in male germ cells as they progress through spermatogenesis. In this study, we evaluated the effects of aging and removing SOD1 (in male germ cells of SOD1-null (Sod1-/-) mice) in order to determine the potential mechanism(s) of DNA damage in these cells. Immunohistochemical analysis showed an increase in lipid peroxidation and DNA damage in the germ cells of aged wild-type (WT) and Sod1-/- mice of all age. Immunostaining of 8-oxoguanine DNA glycosylase, a marker of base excision repair (BER), increased in aged WT and young Sod1-/- mice. In contrast, immunostaining intensity of DNA ligase 4 and RAD51 Recombinase, markers of nonhomologous end-joining (NHEJ), and homologous recombination (HR), respectively, decreased in aged and Sod1-/- mice. Gene expression analysis showed similar results with altered mRNA expression of these key DNA repair transcripts in pachytene spermatocytes and round spermatids of aged and Sod1-/- mice. Our study indicates that DNA repair pathway markers of BER, NHEJ, and HR are differentially regulated as a function of aging and oxidative stress in spermatocytes and spermatids, and aging enhances the repair response to increased oxidative DNA damage, whereas impairments in other DNA repair mechanisms may contribute to the increase in DNA damage caused by aging and the loss of SOD1.

Effects of Aging and Oxidative Stress on Spermatozoa of Superoxide-Dismutase 1- and Catalase-Null Mice.

Advanced paternal age is linked to complications in pregnancy and genetic diseases in offspring. Aging results in excess reactive oxygen species (ROS) and DNA damage in spermatozoa; this damage can be transmitted to progeny with detrimental consequences. Although there is a loss of antioxidants with aging, the impact on aging male germ cells of the complete absence of either catalase (CAT) or superoxide dismutase 1 (SOD1) has not been investigated. We used CAT-null (Cat(-/-)) and SOD1-null (Sod(-/-)) mice to determine whether loss of these antioxidants increases germ cell susceptibility to redox dysfunction with aging. Aging reduced fertility and the numbers of Sertoli and germ cells in all mice. Aged Sod(-/-) mice displayed an increased loss of fertility compared to aged wild-type mice. Treatment with the pro-oxidant SIN-10 increased ROS in spermatocytes of aged wild-type and Sod(-/-) mice, while aged Cat(-/-) mice were able to neutralize this ROS. The antioxidant peroxiredoxin 1 (PRDX1) increased with age in wild-type and Cat(-/-) mice but was consistently low in young and aged Sod(-/-) mice. DNA damage and repair markers (γ-H2AX and 53BP1) were reduced with aging and lower in young Sod(-/-) and Cat(-/-) mice. Colocalization of γ-H2AX and 53BP1 suggested active repair in young wild-type mice but reduced in young Cat(-/-) and in Sod(-/-) mice and with age. Oxidative DNA damage (8-oxodG) increased in young Sod(-/-) mice and with age in all mice. These studies show that aged Sod(-/-) mice display severe redox dysfunction, while wild-type and Cat(-/-) mice have compensatory mechanisms to partially alleviate oxidative stress and reduce age-related DNA damage in spermatozoa. Thus, SOD1 but not CAT is critical to the maintenance of germ cell quality with aging.

Zinc Transport Differs in Rat Spermatogenic Cell Types and Is Affected by Treatment with Cyclophosphamide.

Adequate zinc levels are required for proper cellular functions and for male germ cell development. Zinc transport is accomplished by two families of zinc transporters, the ZIPs and the ZnTs, that increase and decrease cytosolic zinc levels, respectively. However, very little is known about zinc transport in the testis. Furthermore, whether cytotoxic agents such as cyclophosphamide (CPA), a known male germ cell toxicant, can affect zinc transport and homeostasis is unknown. We examined zinc transporter expression and zinc transport in pachytene spermatocytes (PS) and round spermatids (RS) in a normal state and after exposure to CPA. We observed differences in the expression of members of the ZnT and ZIP families in purified populations of PS and RS. We also observed that RS accumulate more zinc over time than PS. The expression of many zinc binding genes was altered after CPA treatment. Interestingly, we found that the expression levels of ZIP5 and ZIP14 were increased in PS from animals treated daily with 6 mg/kg CPA for 4 wk but not in RS. This up-regulation led to an increase in zinc uptake in PS but not in RS from treated animals compared to controls. These data suggest that CPA treatment may alter zinc homeostasis in male germ cells leading to an increased need for zinc. Altered zinc homeostasis may disrupt proper germ cell development and contribute to infertility and effects on progeny.

The Effects of Chemotherapeutic Agents, Bleomycin, Etoposide, and Cisplatin, on Chromatin Remodeling in Male Rat Germ Cells.

The coadministration of bleomycin, etoposide, and cisplatin (BEP) has increased the survival rate of testicular cancer patients to over 90%. Previous studies have demonstrated that BEP induces germ cell damage during the final stages of spermatogenesis, when major chromatin remodeling occurs. Chromatin remodeling permits histone-protamine exchange, resulting in sperm head chromatin compaction. This process involves different epigenetic modifications of the core histones. The objective of these studies was to investigate the effects of BEP on epigenetic modifications to histones involved in chromatin remodeling. Brown Norway rats were treated with BEP, and their testes were removed to isolate pachytene spermatocytes and round spermatids by unit gravity sedimentation. Western blot analyses were conducted on extracted proteins to detect the expression of key modified histones. In a second cohort testes were prepared for immunohistochemical analysis. The stage-specific expression of each modified histone mark in rat spermatogenesis suggests the involvement of these modifications in chromatin remodeling. BEP treatment significantly increased expression of H3K9m and decreased that of tH2B (or Hist1h2ba) in pachytene spermatocytes, suggesting that nucleosomes were not destabilized to allow for transcription of genes involved in chromatin remodeling. Moreover, BEP treatment altered the expression of H4K8ac in round and elongating spermatids, suggesting that histone eviction was compromised, leading to a looser chromatin structure in mature spermatozoa. Less-compacted sperm chromatin, with alterations to the sperm epigenome, may have an adverse effect on male fertility.

Exposure of Female Rats to an Environmentally Relevant Mixture of Brominated Flame Retardants Targets the Ovary, Affecting Folliculogenesis and Steroidogenesis.

Brominated flame retardants (BFRs) are incorporated into various consumer products to prevent flame propagation. These compounds leach into the domestic environment, resulting in chronic exposure and contamination. Pregnancy failure is associated with high levels of BFRs in human follicular fluid, raising serious questions regarding their impact on female reproductive health. The goal of this study is to elucidate the effects of an environmentally relevant BFR mixture on female rat ovarian functions (i.e., folliculogenesis and steroidogenesis). A BFR dietary mixture formulated to mimic the relative BFR congener levels in North American house dust was administered to adult female Sprague-Dawley rats from 2 to 3 wk before mating until Gestational Day 20; these diets were designed to deliver nominal doses of 0, 0.06, 20, or 60 mg/kg/day of the BFR mixture. Exposure to BFRs triggered an approximately 50% increase in the numbers of preantral and antral follicles and an enlargement of the antral follicles in the ovaries of the dams. A significant reduction in the expression of catalase, an antioxidant enzyme, and downregulation of the expression of insulin-like factor 3 (Insl3) and 17alpha-hydroxylase (Cyp17a1) were observed in the ovary. In addition, BFR exposure affected steroidogenesis; we observed a significant decrease in circulating 17-hydroxypregnenolone and an increase in testosterone concentrations in BFR-exposed dams. Thus, BFRs target ovarian function in the rat, adversely affecting both folliculogenesis and steroidogenesis.

HT-COMET: a novel automated approach for high throughput assessment of human sperm chromatin quality.

STUDY QUESTION: Can we make the comet assay (single-cell gel electrophoresis) for human sperm a more accurate and informative high throughput assay? SUMMARY ANSWER: We developed a standardized automated high throughput comet (HT-COMET) assay for human sperm that improves its accuracy and efficiency, and could be of prognostic value to patients in the fertility clinic. WHAT IS KNOWN ALREADY: The comet assay involves the collection of data on sperm DNA damage at the level of the single cell, allowing the use of samples from severe oligozoospermic patients. However, this makes comet scoring a low throughput procedure that renders large cohort analyses tedious. Furthermore, the comet assay comes with an inherent vulnerability to variability. Our objective is to develop an automated high throughput comet assay for human sperm that will increase both its accuracy and efficiency. STUDY DESIGN, SIZE, DURATION: The study comprised two distinct components: a HT-COMET technical optimization section based on control versus DNAse treatment analyses ( ITALIC! n = 3-5), and a cross-sectional study on 123 men presenting to a reproductive center with sperm concentrations categorized as severe oligozoospermia, oligozoospermia or normozoospermia. PARTICIPANTS/MATERIALS, SETTING, METHODS: Sperm chromatin quality was measured using the comet assay: on classic 2-well slides for software comparison; on 96-well slides for HT-COMET optimization; after exposure to various concentrations of a damage-inducing agent, DNAse, using HT-COMET; on 123 subjects with different sperm concentrations using HT-COMET. Data from the 123 subjects were correlated to classic semen quality parameters and plotted as single-cell data in individual DNA damage profiles. MAIN RESULTS AND THE ROLE OF CHANCE: We have developed a standard automated HT-COMET procedure for human sperm. It includes automated scoring of comets by a fully integrated high content screening setup that compares well with the most commonly used semi-manual analysis software. Using this method, a cross-sectional study on 123 men showed no significant correlation between sperm concentration and sperm DNA damage, confirming the existence of hidden chromatin damage in men with apparently normal semen characteristics, and a significant correlation between percentage DNA in the tail and percentage of progressively motile spermatozoa. Finally, the use of DNA damage profiles helped to distinguish subjects between and within sperm concentration categories, and allowed a determination of the proportion of highly damaged cells. LIMITATIONS, REASONS FOR CAUTION: The main limitations of the HT-COMET are the high, yet indispensable, investment in an automated liquid handling system and heating block to ensure accuracy, and the availability of an automated plate reading microscope and analysis software. WIDER IMPLICATIONS OF THE FINDINGS: This standardized HT-COMET assay offers many advantages, including higher accuracy and evenness due to automation of sensitive steps, a 14.4-fold increase in sample analysis capacity, and an imaging and scoring time of 1 min/well. Overall, HT-COMET offers a decrease in total experimental time of more than 90%. Hence, this assay constitutes a more efficient option to assess sperm chromatin quality, paves the way to using this assay to screen large cohorts, and holds prognostic value for infertile patients. STUDY FUNDING/COMPETING INTERESTS: Funded by the CIHR Institute of Human Development, Child and Youth Health (IHDCYH; RHF 100625). O.A. is a fellow supported by the Fonds de la Recherche du Québec - Santé (FRQS) and the CIHR Training Program in Reproduction, Early Development, and the Impact on Health (REDIH). B.R. is a James McGill Professor. The authors declare no conflicts of interest.

Gestational and Early Postnatal Exposure to an Environmentally Relevant Mixture of Brominated Flame Retardants: General Toxicity and Skeletal Variations.

Brominated flame retardants (BFRs) are stable environmental contaminants known to exert endocrine-disrupting effects. Developmental exposure to polybrominated diphenyl ethers (PBDEs) is correlated with impaired thyroid hormone signaling, as well as estrogenic and anti-androgenic effects. As previous studies have focused on a single congener or technical mixture, the purpose of the current study was to examine the effects of gestational and early postnatal exposure to an environmentally relevant mixture of BFRs designed to reflect house dust levels of PBDEs and hexabromocyclododecane on postnatal developmental outcomes. Pregnant Sprague-Dawley rats were exposed to the PBDE mixture from preconception to weaning (PND 21) through the diet containing 0, 0.75, 250, and 750 mg mixture/kg diet. BFR exposure induced transient reductions in body weight at PND 35 in male and from PND 30-45 in female offspring (250 and 750 mg/kg). Liver weights (PND 21) and xenobiotic metabolizing enzyme activities (PND 21 and 46) were increased in both male and female offspring exposed to 250 and 750 mg/kg diets. Furthermore, serum T4 levels were reduced at PND 21 in both,male and female offspring (250 and 750 mg/kg). At PND 21, Serum alkaline phosphatase (ALP) was decreased in males exposed to 750 mg/kg dietat, and females exposed to 250 and 750 mg/kg diets. At PND 46 ALP was significantly elevated in males (250 and 750 mg/kg). Variations in the cervical vertebrae and phalanges were observed in pups at PND 4 (250 and 750 mg/kg). Therefore, BFR exposure during gestation through to weaning alters developmental programming in the offspring. The persistence of BFRs in the environment remains a cause for concern with regards to developmental toxicity.

Male Rat Germ Cells Display Age-Dependent and Cell-Specific Susceptibility in Response to Oxidative Stress Challenges.

For decades male germ cells were considered unaffected by aging, due to the fact that males continue to generate sperm into old age; however, evidence indicates that germ cells from aged males are of lower quality than those of young males. The current study examines the effects of aging on pachytene spermatocytes and round spermatids, and is the first study to culture these cells in isolation for an extended period. Our objective is to determine the cell-specific responses germ cells have to aging and oxidative insult. Culturing isolated germ cells from young and aged Brown Norway rats revealed that germ cells from aged males displayed an earlier decline in viability, elevated levels of reactive oxygen species (ROS), and increased spermatocyte DNA damage, compared to young males. Furthermore, oxidative insult by prooxidant 3-morpholinosydnonimine provides insight into how spermatocytes and spermatids manage excess ROS. Genome-wide microarray analyses revealed that several transcripts for antioxidants, Sod1, Cat, and Prdxs, were up-regulated in response to ROS in germ cells from young males while being expressed at lower levels in the aged. In contrast, the expression of DNA damage repair genes Rad50 and Atm were increased in the germ cells from aged animals. Our data indicate that as germ cells undergo spermatogenesis, they adapt and respond to oxidative stress differently, depending on their phase of development, and the process of aging results in redox dysfunction. Thus, even at early stages of spermatogenesis, germ cells from aged males are unable to mount an appropriate response to manage oxidative stress.

Effects of four chemotherapeutic agents, bleomycin, etoposide, cisplatin, and cyclophosphamide, on DNA damage and telomeres in a mouse spermatogonial cell line.

Treatment with chemotherapeutics agents may induce persistent DNA damage in male germ cells with the possibility of long-term consequences on fertility and progeny outcome. Telomeres, specialized structures at the physical ends of chromosomes, play an important role in the maintenance of genetic stability and in the response of somatic cells to anticancer drugs. Our objective was to test the hypothesis that exposure to bleomycin, etoposide, or cisplatin (the drugs used to treat testicular cancer) or cyclophosphamide (an anticancer agent and immunosuppressant) targets telomeres in the male germ line. C18-4 spermatogonial cells were exposed to bleomycin, etoposide, cisplatin, or 4-hydroperoxycyclophosphamide (4OOH-CPA, a preactivated analog of cyclophosphamide). All four anticancer drugs induced a significant increase in DNA damage in C18-4 cells, as assessed by gamma-H2AX immunofluorescence. Interestingly, the gamma-H2AX signal was localized to telomeres after treatment with bleomycin, cisplatin, and 4OOH-CPA, but not etoposide. Mean telomere lengths, the intensity of the telomere fluorescence in situ hybridization signal, telomerase activity, and the expression of the telomerase enzyme mRNA components, Tert and Terc, were reduced by exposure to cisplatin and 4OOH-CPA, but not by bleomycin or etoposide. Thus, although all four anticancer drugs induced DNA damage in this spermatogonial cell line, telomeres were not specifically affected by etoposide and only the two alkylating agents, cisplatin and 4OOH-CPA, induced telomere dysfunction. This telomere dysfunction may contribute to infertility and developmental defects in the offspring.

Hair as a biomarker of systemic exposure to polybrominated diphenyl ethers.

The efficacy of using hair as a biomarker for exposure to polybrominated diphenyl ether (PBDE) flame retardants was assessed in humans and an animal model. Paired human hair and serum samples were obtained from adult men and women (n = 50). In parallel, hair, serum, liver, and fat were collected from adult male Sprague-Dawley rats exposed to increasing doses of the PBDE mixture found in house dust for 70 days via the diet. All samples were analyzed by GC-MS for eight common PBDEs: BDE-28, -47, -99, -100, -153, -154, -183, and -209. Paired human hair and serum samples had five congeners (BDE-28, -47, -99, -100, and -154) with significant individual correlations (0.345-0.566). In rat samples, BDE-28 and BDE-183 were frequently below the level of detection. Significant correlations were observed for BDE-47, -99, -100, -153, -154, and -209 in rat hair, serum, liver, and fat across doses, with r values ranging from 0.803 to 0.988; weaker correlations were observed between hair and other tissues when data from the lowest dose group or for BDE-209 were analyzed. Thus, human and rat hair PBDE measurements correlate strongly with those in alternative matrices, validating the use of hair as a noninvasive biomarker of long-term PBDE exposure.

Impact of Exposure to a Mixture of Organophosphate Esters on Adrenal Cell Phenotype, Lipidome, and Function.

Organophosphate esters (OPEs) are used primarily as flame retardants and plasticizers. Previously, we reported that adrenal cells are important targets of individual OPEs. However, real-life exposures are to complex mixtures of these chemicals. To address this, we exposed H295R human adrenal cells to varying dilutions (1/1000K to 1/3K) of a Canadian household dust-based OPE mixture for 48 hours and evaluated effects on phenotypic, lipidomic, and functional parameters. Using a high-content screening approach, we assessed phenotypic markers at mixture concentrations at which there was greater than 70% cell survival; the most striking effect of the OPE mixture was a 2.5-fold increase in the total area of lipid droplets. We then determined the response of specific lipid species to OPE exposures with novel, nontargeted lipidomic analysis of isolated lipid droplets. These data revealed that house dust OPEs induced concentration-dependent alterations in the composition of lipid droplets, particularly affecting the triglyceride, diglyceride, phosphatidylcholine, and cholesterol ester subclasses. The steroid-producing function of adrenal cells in the presence or absence of a steroidogenic stimulus, forskolin, was determined. While the production of 17ß-estradiol remained unaffected, a slight decrease in testosterone production was observed after stimulation. Conversely, a 2-fold increase in both basal and stimulated cortisol and aldosterone production was observed. Thus, exposure to a house dust-based mixture of OPEs exerts endocrine-disrupting effects on adrenal cells, highlighting the importance of assessing the effects of environmentally relevant mixtures.

Organophosphate ester flame retardants and plasticizers affect the phenotype and function of HepG2 liver cells.

Exposure to the organophosphate esters (OPEs), used as flame retardants and plasticizers, is associated with a variety of adverse health effects including an increase in the incidence of fatty liver diseases. The goal of this study was to investigate the effects of six OPEs, all detected in Canadian house dust, on the phenotype and function of HepG2 liver cells. We used high-content imaging to investigate the effects of these OPEs on cell survival, mitochondria, oxidative stress, lipid droplets, and lysosomes. Effects on the autophagy/lipophagy pathway were evaluated using confocal microscopy. The triaryl OPEs (isopropylated triphenylphosphate [IPPP], tris(methylphenyl) phosphate [TMPP], and triphenyl phosphate [TPHP]) were more cytotoxic than non-triaryl OPEs (tris(2-butoxyethyl) phosphate [TBOEP], tris(1-chloro-2-propyl) phosphate [TCIPP], and tris(1,3-dichloro-2-propyl) phosphate [TDCIPP]). Exposure to most OPEs increased total mitochondria, reduced reactive oxygen species, and increased total lipid droplet areas and lysosomal intensity. Potency ranking was done using the lowest benchmark concentration/administered equivalent dose method and toxicological prioritization index analyses to integrate all phenotypic endpoints. IPPP, TBOEP, and TPHP ranked as the most potent OPEs, whereas TMPP, TCIPP, and TDCIPP were relatively less bioactive. Confocal microscopic analysis demonstrated that IPPP reduced the colocalization of lipid droplets (PLIN2), lysosomes (LAMP1), and autophagosomes (p62), disrupting autophagy. In contrast, TBOEP rescued cells from bafilomycin A1-induced inhibition of autophagy and/or increased autophagic flux. Together, these data demonstrate that OPEs have adverse effects on HepG2 cells. Further, OPE-induced dysregulation of autophagy may contribute to the association between OPE exposure and adverse effects on liver lipid homeostasis.

House dust-derived mixtures of organophosphate esters alter the phenotype, function, transcriptome, and lipidome of KGN human ovarian granulosa cells.

Organophosphate esters (OPEs), used as flame retardants and plasticizers, are present ubiquitously in the environment. Previous studies suggest that exposure to OPEs is detrimental to female fertility in humans. However, no experimental information is available on the effects of OPE mixtures on ovarian granulosa cells, which play essential roles in female reproduction. We used high-content imaging to investigate the effects of environmentally relevant OPE mixtures on KGN human granulosa cell phenotypes. Perturbations to steroidogenesis were assessed using ELISA and qRT-PCR. A high-throughput transcriptomic approach, TempO-Seq, was used to identify transcriptional changes in a targeted panel of genes. Effects on lipid homeostasis were explored using a cholesterol assay and global lipidomic profiling. OPE mixtures altered multiple phenotypic features of KGN cells, with triaryl OPEs in the mixture showing higher potencies than other mixture components. The mixtures increased basal production of steroid hormones; this was mediated by significant changes in the expression of critical transcripts involved in steroidogenesis. Further, the total-OPE mixture disrupted cholesterol homeostasis and the composition of intracellular lipid droplets. Exposure to complex mixtures of OPEs, similar to those found in house dust, may adversely affect female reproductive health by altering a multitude of phenotypic and functional endpoints in granulosa cells. This study provides novel insights into the mechanisms of actions underlying the toxicity induced by OPEs and highlights the need to examine the effects of human relevant chemical mixtures.

Aging results in molecular changes in an enriched population of undifferentiated rat spermatogonia.

A strong correlation exists between increasing paternal age and a decline in reproductive function. Testis aging is associated with testicular atrophy, increased DNA damage, and de novo mutations. It is unclear whether these problems arise from the spermatogonial stem cells (SSCs), a buildup of anomalies as older germ cells progress through spermatogenesis, or both. We hypothesize that with the continual divisions of SSCs that maintain the germ cell population, an alteration of these cells occurs over time. To test this, we utilized young (4-mo-old) and aged (18- and 21-mo-old) transgenic rats that express GFP in germ cells only. We first examined the number and activity of SSCs from the different age groups by transplantation. Aged rats had numerically fewer SSCs than young rats (<50%; not significant) despite the lack of testicular atrophy, and 21-mo-old rats show a significant reduction in colony length, suggesting that the quality of SSCs also deteriorates. To evaluate any molecular changes occurring in the early cells of spermatogenesis with age, we isolated an SSC-enriched population of CD9-positive (CD9(+)) cells using fluorescence-activated cell sorting (confirmed by transplantation studies) and extracted RNA for microarray analysis. In the aged CD9(+) cells, 60 transcripts were upregulated and more than 500 downregulated compared to the young cells. An altered expression was found for transcripts involved in mitosis and in DNA damage response. These results suggest molecular alterations in the SSC-enriched population of aged CD9(+) cells, implying that reproductive aging originates in the undifferentiated cells of spermatogenesis.

The effects of chemotherapy with bleomycin, etoposide, and cis-platinum (BEP) on rat sperm chromatin remodeling, fecundity and testicular gene expression in the progeny.

During spermiogenesis, histones are replaced first by transition proteins and then by protamines, resulting in a very condensed sperm DNA structure that is absolutely critical for normal sperm function. We have demonstrated previously that, despite a 9-wk recovery period, mature sperm from rats treated for 9 wk with bleomycin, etoposide, and cis-platinum (BEP), the drugs used to treat testicular cancer, have reduced levels of protamine 1 and a concomitant upregulation of specific histones, highlighting a problem in histone eviction. Here, we demonstrate that regulators of histone removal are increased in elongating spermatids following recovery; however, Ac-H4 and gamma H2AX histones remain elevated in elongating spermatids or caudal epididymal spermatozoa 9 wk post-BEP treatment. This indicates that chromatin remodelers and effector proteins that respond to histone removal cues may be a target of BEP treatment. A decrease in the expression of SMARCE1 in elongating spermatids may explain the persistent retention of histones in cauda epididymal sperm 9 wk after the cessation of BEP treatment. Remarkably, proteins implicated in the translational control and posttranslational processing of protamine 1 are also significantly elevated 9 wk post-BEP treatment, suggesting that histone eviction may dictate the DNA availability for protamine binding. Males mated to control females 9 wk after BEP treatment have reduced litter sizes; moreover, the profile of gene expression in the developing testes of their pups is altered. Altering the proportion of histones to protamine in mature spermatozoa has an adverse impact on male fecundity, with modifications to epigenetic marks potentially threatening normal progeny development.