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.

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 non-alcoholic fatty liver disease. 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(2-butoxyethyl) phosphate [TBOEP], and triphenyl phosphate [TPHP]) were more cytotoxic than non-triaryl OPEs (tris(methylphenyl) phosphate [TMPP], tris(1-chloro-2-propyl) phosphate [TCIPP], and tris(1,3-dichloro-2-propyl) phosphate [TDCIPP]). Exposure to most OPEs increased Mitotracker green intensity and reduced reactive oxygen species, 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 co-localization 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.

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.

Toxicological Mechanisms and Potencies of Organophosphate Esters in KGN Human Ovarian Granulosa Cells as Revealed by High-throughput Transcriptomics.

Despite the growing number of studies reporting potential risks associated with exposure to organophosphate esters (OPEs), their molecular mechanisms of action remain poorly defined. We used the high-throughput TempO-Seq™ platform to investigate the effects of frequently detected OPEs on the expression of ∼3000 environmentally responsive genes in KGN human ovarian granulosa cells. Cells were exposed for 48 h to one of five OPEs (0.1 to 50 µM): tris(methylphenyl) phosphate (TMPP), isopropylated triphenyl phosphate (IPPP), tert-butylphenyl diphenyl phosphate (BPDP), triphenyl phosphate (TPHP), or tris(2-butoxyethyl) phosphate (TBOEP). The sequencing data indicate that four OPEs induced transcriptional changes, whereas TBOEP had no effect within the concentration range tested. Multiple pathway databases were used to predict alterations in biological processes based on differentially expressed genes. At lower concentrations, inhibition of the cholesterol biosynthetic pathway was the predominant effect of OPEs; this was likely a consequence of intracellular cholesterol accumulation. At higher concentrations, BPDP and TPHP had distinct effects, primarily affecting pathways involved in cell cycle progression and other stress responses. Benchmark concentration (BMC) modelling revealed that BPDP had the lowest transcriptomic point of departure. However, in vitro to in vivo extrapolation modeling indicated that TMPP was bioactive at lower concentrations than the other OPEs. We conclude that these new approach methodologies provide information on the mechanism(s) underlying the effects of data-poor compounds and assist in the derivation of protective points of departure for use in chemical read-across and decision-making.

The Organophosphate Esters Used as Flame Retardants and Plasticizers Affect H295R Adrenal Cell Phenotypes and Functions.

Adverse effects associated with exposure to brominated flame retardants have led to regulations for their use and their replacement with organophosphate esters (OPEs). However, little is known about the impact of OPEs on the adrenal, a vital endocrine gland. Here, we used a high-content screening approach to elucidate the effects of OPEs on H295R human adrenal cell phenotypic endpoints and function. The effects of 2,2',4,4'-tetrabromodiphenyl ether (BDE-47), a legacy brominated flame retardant, on H295R cell cytotoxicity, oxidative stress, mitochondria, lysosomes, and lipid droplets were compared with those of 6 OPEs. Most OPEs reduced oxidative stress, increased the numbers of mitochondria, decreased lysosomes, and increased lipid droplets. Two potency ranking approaches, the lowest benchmark concentration/administered equivalent dose methods and Toxicological Prioritization Index analyses, revealed that the triaryl-OPEs (isopropylated triphenyl phosphate [IPPP], tris(methylphenyl) phosphate [TMPP], and triphenyl phosphate [TPHP]) and 1 nontriaryl OPE (tris(1,3-dichloro-2-propyl) phosphate [TDCIPP]) were more potent than BDE-47. The steroidogenic activity of adrenal cells in the presence or absence of forskolin, a steroidogenic stimulus, was determined after exposure to triaryl-OPEs. The basal production of cortisol and aldosterone was increased by IPPP but decreased by TPHP or TMPP exposure; the response to forskolin was not affected by these OPEs. All 3 triaryl OPEs altered the expression of rate-limiting enzymes involved in cholesterol and steroid biosynthesis; CYP11B1 and CYP11B2 were the most prominently affected targets. The OPE chemical-specific effects on cortisol and aldosterone production were best explained by alterations in STAR expression. Thus, the adrenal may be an important target for these endocrine-disrupting chemicals.

Organophosphate Esters Disrupt Steroidogenesis in KGN Human Ovarian Granulosa Cells.

Organophosphate esters (OPEs) are used extensively as flame retardants and plasticizers and are found ubiquitously in the environment and human matrices. Previous studies suggested that exposure to some of these chemicals may disrupt the homeostasis of female sex hormones and have detrimental effects on female fertility. Here, we determined the effects of OPEs on the function of KGN ovarian granulosa cells. We hypothesized that OPEs alter the steroidogenic ability of these cells by dysregulating the expression of transcripts involved in steroid and cholesterol biosynthesis. KGN cells were exposed for 48 hours to 1 of 5 OPEs (1-50µM): triphenyl phosphate (TPHP), tris(methylphenyl) phosphate (TMPP), isopropylated triphenyl phosphate (IPPP), tert-butylphenyl diphenyl phosphate (BPDP), and tributoxyethyl phosphate (TBOEP), or to a polybrominated diphenyl ether flame retardant, 2,2',4,4' tetrabromodiphenyl ether (BDE-47), in the presence or absence of Bu2cAMP. OPEs increased the basal production of progesterone (P4) and 17ß-estradiol (E2) and had either no effect or inhibited Bu2cAMP-stimulated P4 and E2 synthesis; exposure to BDE-47 had no effect. Quantitative real-time polymerase chain reaction (qRT-PCR) analyses revealed that OPEs (≥5µM) increased the basal expression of critical genes (STAR, CYP11A1, CYP19A1, HSD3B2, and NR5A1) involved in steroidogenesis; upon stimulation, the expression of all genes tested was downregulated. An overall inhibition in cholesterol biosynthesis was induced by OPEs, characterized by a downregulation in HMGCR and SREBF2 expression. TBOEP consistently showed the least effect. Therefore, OPEs perturbed steroidogenesis in KGN granulosa cells by targeting the expression of steroidogenic enzymes and cholesterol transporters; these effects may have an adverse impact on female reproduction.

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.

Influence of androgens on the innate immune system.

BACKGROUND: Sexual dimorphism is observed in the occurrence, course, and severity of human disease. The difference in immune response between males and females can in part be attributed to sexual genotype. However, immunological differences can also be explained by endocrine-immune interactions. Specifically, androgens possess the ability of directly modulating the development and function of immune cells. Although androgens generally contribute to immunosuppressive effects, this is not necessarily always the case. AIM: The aim of the review is to uncover the role of androgens in shaping the innate immune response. MATERIAL & METHODS: Authors included papers in this review which discussed the impact of androgens on specific innate immune cells. RESULTS: Androgens modulate the innate immune response through various mechanisms. However, there is conflicting evidence in the literature regarding the interplay betwen androgens and the innate immune system. DISCUSSION: Conflicting evidence presented in this review could in part be explained by the limitations present in interpreting results. CONCLUSION: This review is of great importance for our understanding of occurence and mechanism of human inflammatory disease.

Sperm DNA methylome abnormalities occur both pre- and post-treatment in men with Hodgkin disease and testicular cancer.

BACKGROUND: Combination chemotherapy has contributed to increased survival from Hodgkin disease (HD) and testicular cancer (TC). However, questions concerning the quality of spermatozoa after treatment have arisen. While studies have shown evidence of DNA damage and aneuploidy in spermatozoa years following anticancer treatment, the sperm epigenome has received little attention. Our objectives here were to determine the impact of HD and TC, as well as their treatments, on sperm DNA methylation. Semen samples were collected from community controls (CC) and from men undergoing treatment for HD or TC, both before initiation of chemotherapy and at multiple times post-treatment. Sperm DNA methylation was assessed using genome-wide and locus-specific approaches. RESULTS: Imprinted gene methylation was not affected in the sperm of HD or TC men, before or after treatment. Prior to treatment, using Illumina HumanMethylation450 BeadChip (450 K) arrays, a subset of 500 probes was able to distinguish sperm samples from TC, HD and CC subjects; differences between groups persisted post-treatment. Comparing altered sperm methylation between HD or TC patients versus CC men, twice as many sites were affected in TC versus HD men; for both groups, the most affected CpGs were hypomethylated. For TC patients, the promoter region of GDF2 contained the largest region of differential methylation. To assess alterations in DNA methylation over time/post-chemotherapy, serial samples from individual patients were compared. With restriction landmark genome scanning and 450 K array analyses, some patients who underwent chemotherapy showed increased alterations in DNA methylation, up to 2 to 3 years post-treatment, when compared to the CC cohort. Similarly, a higher-resolution human sperm-specific assay that includes assessment of environmentally sensitive regions, or "dynamic sites," also demonstrated persistently altered sperm DNA methylation in cancer patients post-treatment and suggested preferential susceptibility of "dynamic" CpG sites. CONCLUSIONS: Distinct sperm DNA methylation signatures were present pre-treatment in men with HD and TC and may help explain increases in birth defects reported in recent clinical studies. Epigenetic defects in spermatozoa of some cancer survivors were evident even up to 2 years post-treatment. Abnormalities in the sperm epigenome both pre- and post-chemotherapy may contribute to detrimental effects on future reproductive health.

Aging affects gene expression in spermatids of Brown Norway rats.

The effects of aging on the reproductive health of men and the consequences for their offspring are becoming more widely recognized. Correlative epidemiological studies examining paternal age and offspring health suggest there are more frequent occurrences of genetic disorders in the children of older fathers. Given the genetic basis for paternal age-related disorders, we aim to characterize gene expression in developing germ cells. Round spermatids (RS) were collected from young (mean = 5.3 months) and aged (mean = 19.5 months) Brown Norway rats, representative of humans aged 20-30 years and 55+ years, respectively. Gene expression data were obtained by mRNA sequencing (n = 5), and were analysed for differential expression. Sequencing data display 211 upregulated and 9 downregulated transcripts in RS of aged rats, compared to young (log2FC >1, p < 0.05). Transcripts with increased expression are involved in several processes including sperm motility/morphology, sperm-egg binding, capacitation, and epigenetic inheritance. In addition, there are numerous dysregulated transcripts that regulate germ cell epigenetic marks and Sertoli-germ cell binding and communication. These results show an overall increase in RS gene expression with age, with spermatogenic functions being perturbed. Taken together, these findings help identify the genetic origin of the fertility, germ cell niche, and epigenetic effects observed with advanced paternal aging.

Gestational and Lactational Exposure to the Emergent Alternative Plasticizer 1,2-Cyclohexane Dicarboxylic Acid Diisononyl Ester (DINCH) Impairs Lipid Metabolism to a Greater Extent Than the Commonly Used Di(2-Ethylhexyl) Phthalate (DEHP) in the Adult Rat Mammary Gland.

Due to their endocrine disruption properties, phthalate plasticizers such as di(2-ethylhexyl) phthalate (DEHP) can affect the hormone-dependent development of the mammary gland. Over the past few years, DEHP has been partially replaced by 1,2-cyclohexane dicarboxylic acid diisononyl ester (DINCH) which also have potential endocrine disrupting properties. The goal of the present study is to understand the impact of a gestational and lactational exposure to DEHP and DINCH on mammary gland development using Sprague Dawley rats. Both plasticizers altered the adipocytes of the mammary gland fat pad of adult progeny, as demonstrated by a decrease in their size, folding of their membrane, and modulations of the lipid profiles. DEHP treatments decreased the expression of Rxrα and Scd1 at the low and high dose, respectively, but did not affect any of the other genes studied. DINCH modulation of lipid metabolism could be observed at puberty by a decreased expression of genes implicated in triglyceride synthesis, lipid transport, and lipolysis, but by an increased expression of genes of the ß-oxidation pathway and of genes involved in lipid storage and fatty acid synthesis at adulthood, compared with control and DEHP-treated rats. A strong upregulation of different inflammatory markers was observed following DINCH exposure only. Together, our results indicate that a gestational and lactational exposure to DINCH has earlier and more significant effects on lipid homeostasis, adipogenesis, and the inflammatory state of the adult mammary gland than DEHP exposure. The long-term consequence of these effects on mammary gland health remained to be determined.

Exposure of men living in the greater Montreal area to organophosphate esters: Association with hormonal balance and semen quality.

Exposure to organophosphate esters (OPEs) is extensive, yet few studies have investigated their association with hormone levels or semen quality. Here, we studied the association between urinary concentrations of OPEs and their metabolites with hormone levels and semen parameters in men (n = 117) predominantly in the 20-29 years age range who were recruited from the greater Montreal area between 2009 and 2012. Urine, serum, and semen samples were analyzed for OPEs, hormones, and semen quality, respectively. Bis(2-ethylhexyl) phosphate (BEHP), bis(2,4-di-tert-butylphenyl) hydrogen phosphate (B2,4DtBPP), tris(2-chloroisopropyl) phosphate (TCIPP), diphenyl phosphate (DPHP), bis (2-butoxyethyl) phosphate (BBOEP) and di-cresyl phosphate (DCPs) were detected in urine at a frequency ≥ 95%. The highest geometric mean concentration was observed for DPHP (18.54 ng/mL) and the second highest was B2,4DtBPP (6.23 ng/mL). Associations between a doubling in analyte concentrations in urine and hormone levels and semen quality parameters were estimated using multivariable linear regression. B2,4DtBPP levels were positively associated with total T3 (ß = 0.09; 95% CI: 0.01, 0.17). DPHP was inversely associated with estradiol (ß = -2.56; 95% CI: -5.00, -0.17), and TCIPP was inversely associated with testosterone (ß = -0.78; 95% CI: -1.40, -0.17). Concentrations of BCIPP were inversely associated with sperm concentrations (ß = -7.76; 95% CI: -14.40, -0.61), progressive motility (ß = - 4.98; 95% CI: -8.71, -1.09), and the sperm motility index (ß = -9.72; 95% CI: -17.71, -0.96). In contrast, urinary DPHP concentrations were positively associated with the sperm motility (ß = 4.37; 95% CI: 0.76, 8.12) and fertility indices (ß = 6.64; 95% CI: 1.96, 11.53). Thus, OPE detection rates were high and exposure to several OPEs was associated with altered hormone levels and semen parameters. The possibility that OPEs affect male reproduction warrants further investigation.

Advanced Paternal Age and Future Generations.

Paternal age at conception has been increasing. In this review, we first present the results from the major mammalian animal models used to establish that increasing paternal age does affect progeny outcome. These models provide several major advantages including the possibility to assess multi- transgenerational effects of paternal age on progeny in a relatively short time window. We then present the clinical observations relating advanced paternal age to fertility and effects on offspring with respect to perinatal health, cancer risk, genetic diseases, and neurodevelopmental effects. An overview of the potential mechanism operating in altering germ cells in advanced age is presented. This is followed by an analysis of the current state of management of reproductive risks associated with advanced paternal age. The numerous challenges associated with developing effective, practical strategies to mitigate the impact of advanced paternal age are outlined along with an approach on how to move forward with this important clinical quandary.

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.

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.

The Effects of Organophosphate Esters Used as Flame Retardants and Plasticizers on Granulosa, Leydig, and Spermatogonial Cells Analyzed Using High-Content Imaging.

The replacement of regulated brominated flame retardants and plasticizers with organophosphate esters (OPEs) has led to their pervasive presence in the environment and in biological matrices. Further, there is evidence that exposure to some of these chemicals is associated with reproductive toxicity. Using a high-content imaging approach, we assessed the effects of exposure to 9 OPEs on cells related to reproductive function: KGN human granulosa cells, MA-10 mouse Leydig cells, and C18-4 mouse spermatogonial cells. The effects of OPEs were compared with those of 2,2',4,4'-tetrabromodiphenyl ether (BDE-47), a legacy brominated flame retardant. Alterations in several important cell features, including cell survival, mitochondrial dynamics, oxidative stress, lysosomes, and lipid droplets, were analyzed. Most of the OPEs tested displayed higher cytotoxicity than BDE-47 in all 3 cell lines. Effects on phenotypic parameters were specific for each cell type. Several OPEs increased total mitochondria, decreased lysosomes, increased the total area of lipid droplets, and induced oxidative stress in KGN cells; these endpoints were differentially affected in MA-10 and C18-4 cells. Alterations in cell phenotypes were highly correlated in the 2 steroidogenic cell lines for a few triaryl OPEs. Potency ranking using 2 complementary approaches, Toxicological Prioritization Index analyses and the lowest benchmark concentration/administered equivalent dose method, revealed that while most of the OPEs tested were more potent than BDE-47, others showed little to no effect. We propose that these approaches serve as lines of evidence in a screening strategy to identify the potential for reproductive and endocrine effects of emerging chemicals and assist in regulatory decision-making.

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.

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.

Effects of Bisphenols A, AF, and S on Endochondral Ossification and the Transcriptome of Murine Limb Buds.

Bisphenols are a family of chemicals commonly used to produce polycarbonate plastics and epoxy resins. Exposure to bisphenol A (BPA) is associated with a variety of adverse effects; thus, many alternatives to BPA, such as bisphenol AF (BPAF) and bisphenol S (BPS), are now emerging in consumer products. We have determined the effects of 3 bisphenols on endochondral ossification and the transcriptome in a murine limb bud culture system. Embryonic forelimbs were cultured in the presence of vehicle, BPA, BPAF, or BPS. BPA (≥10 µM), BPAF (≥1 µM), and BPS (≥50 µM) reduced the differentiation of hypertrophic chondrocytes and osteoblasts. Chondrogenesis was suppressed by exposure to ≥50 µM BPA, ≥5 µM BPAF, or 100 µM BPS and osteogenesis was almost completely arrested at 100 µM BPA or 10 µM BPAF. RNA sequencing analyses revealed that the total number of differentially expressed genes increased with time and the concentration tested. BPA exposure differentially regulated 635 genes, BPAF affected 554 genes, whereas BPS affected 95 genes. Although the genes that were differentially expressed overlapped extensively, each bisphenol also induced chemical-specific alterations in gene expression. BPA- and BPAF-treated limbs exhibited a downregulation of Rho-specific guanine nucleotide dissociation inhibitor (RhoGDI) signaling genes. Exposure to BPA and BPS resulted in the upregulation of key genes involved in cholesterol biosynthesis, whereas exposure to BPAF induced an upregulation of genes involved in bone formation and in the p53 signaling pathway. These data suggest that BPAF may be more detrimental to endochondral ossification than BPA, whereas BPS is of comparable toxicity to BPA.