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Environ Pollut. 2024 Mar 6;348:123730. doi: 10.1016/j.envpol.2024.123730. Epub ahead of print.
Authors: Chi ZH, Liu L, Zheng J, Tian L, Chevrier J, Bornman R, Obida M, Goodyer CG, Hales BF, Bayen S.

Abstract
A sensitive modified QuEChERS extraction method was developed to assess the levels of free and conjugated bisphenols (BPs) in human milk collected between 2018 and 2019 from two regions of South Africa (the Limpopo Province Vhembe district, n = 194; Pretoria, n = 193) and Canada (Montreal, n = 207). Total BPA (free and conjugated) and BPS were the predominant bisphenols detected in samples from Vhembe and Pretoria, whereas total BPS was the predominant bisphenol detected in Montreal samples. The levels of total BPA in samples from Vhembe and Pretoria ranged between < MDL-18.61 and <MDL-19.38 ng/mL, with medians of 1.03 ng/mL and 0.69 ng/mL and detection frequencies of 73% and 68%, respectively. The speciation analysis of BPA revealed a predominantly conjugated form in South African samples. In contrast, total BPA was detected in only one milk sample from Montreal. Total BPS levels were lower than BPA in South Africa, with detection frequencies of 57% and 21% in Vhembe and Pretoria, respectively. In contrast, total BPS was the major BP detected (42%) in Montreal (up to 4.42 ng/mL). BPAF was found exclusively in South Africa, with detection frequencies for total BPAF of 40% and 9% in Vhembe (<MDL-12.41 ng/mL) and Pretoria (<MDL-0.11 ng/mL), respectively. To our knowledge, this is one of the first studies to detect bisphenols in human milk from data-scarce countries such as South Africa and to highlight the notable disparities in the types and levels of bisphenols detected across two distinct countries (Canada and South Africa).

PMID: 38458524 [PubMed - as supplied by publisher]

Sci Total Environ. 2024 Feb 20:912:169333. doi: 10.1016/j.scitotenv.2023.169333. Epub 2023 Dec 12.
Authors: Struzina L, Pineda M, Yargeau V.

Abstract
The occurrence of thirty-four flame retardants and plasticizers throughout treatment steps in a drinking water treatment plant (DWTP) was analyzed to assess removal efficiencies of filtration, ultraviolet (UV) treatment, and chlorination. Legacy compounds and replacements were included to compare their presence and persistence. Twenty-four-hour composite sampling, offset to account for retention time, was performed at a direct filtration DWTP in Montreal, Canada over a three-day period. Polybrominated diphenyl ethers (PBDEs), considered legacy flame retardants, were infrequently detected or at concentrations <1 ng/L. When overall removal efficiencies could be calculated, the removal of ∑7PBDEs was 49 and 94 % for days 2 and 3, respectively. No removal could be calculated on day 1 as PBDEs were only detected in finished drinking water. Higher brominated PBDEs BDE-183 and BDE-154 were only detected in raw water. Organophosphate esters (OPEs), considered replacement flame retardants, were frequently detected in all water samples. The total average concentration of ∑15OPes was 501 ng/L in raw water and 162 ng/L in drinking water, with an average removal efficiency of 67 %. OPEs were mainly removed during filtration, with TCIPP, TDCIPP, and TPHP showing statistically significant removal of 76, 84, and 95 %, respectively. The total average concentration of ∑8plasticizers was 2938 ng/L in raw water and 116 ng/L in drinking water. All plasticizers, except for metabolite MEHP, had significant removal from filtration, and the overall removal of plasticizers ranged from 20 % for DEP to 99 % for DEHP. Drinking water treatment decreases the concentration of these contaminants in drinking water but was less effective in removing flame retardants than plasticizers, as indicated by their higher number of PBDEs detected and higher concentrations of OPEs measured. To our knowledge, it is the first report of the removal of PBDEs, OPE metabolites and plasticizer replacements (DEHA, DIDA, DINCH, DINP) during drinking water treatment.

PMID: 38097079 [PubMed - as supplied by publisher]

Toxicol Sci. 2023 Nov 6:kfad114. doi: 10.1093/toxsci/kfad114. Epub ahead of print.
Authors: Wang X, Rowan-Carroll A, Meier MJ, Williams A, Yauk CL, Hales BF, Robaire B.

Abstract
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.

PMID: 37941476 [PubMed - as supplied by publisher]

Endocrinology. 2023 Aug 1;164(9):bqad119. doi: 10.1210/endocr/bqad119.
Authors: Li Z, Robaire B, Hales BF.

Abstract
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.

PMID: 37522340 [PubMed - as supplied by publisher]

Endocrinology. 2023 Jun 6;164(7):bqad089. doi: 10.1210/endocr/bqad089.
Authors: Wang X, Lee E, Hales BF, Robaire B.

Abstract
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 17β-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.

PMID: 37288667 [PubMed - as supplied by publisher]

Biol Reprod. 2023 May 10;108(5):837-848. doi: 10.1093/biolre/ioad018.
Authors: Tardif S, Rwigemera A, Letourneau N, Robaire B, Delbes G.

Abstract
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.

PMID: 36780129 [PubMed - as supplied by publisher]

Chemosphere. 2023 341-139908. doi:10.1016/j.chemosphere.2023.139908.Ìý
Authors: Leung G, Akiki C, Bilamjian S, Tian L, Liu L, Bayen S.

Abstract
Flame retardants (FRs) are often added to commercial products to achieve flammability resistance, but they are not chemically bonded to the materials, so, they can be easily released into the environment during the production and disposal processes. When honeybees travel to collect nectar during the pollination process, they are prone to be contaminated by chemicals in the air. Therefore, honey contamination has been proposed as an indicator of the pollution status in a particular region. To date, the occurrence of flame retardants in urban honey has yet to be explored. In this study, a direct injection method was used, coupled with LC-QTOF-MS, to analyze honey samples. This method was applied to urban (n = 100) and rural (n = 100) honey samples from the Quebec province (Canada), and the levels of flame retardants in urban and rural honey samples were not significantly different. In the targeted approach, two of the target FRs, tris(2-butoxyethyl) phosphate (TBOEP) and triphenyl phosphate (TPHP), were detected and confirmed at an average trace concentration (<1 ng mL-1). Additionally, a non-targeted screening workflow with an in-house-built library was developed and validated to screen for flame retardants in honey. Tris (2-chloropropyl) phosphate (TCIPP) was identified in honey using the non-targeted screening workflow and confirmed using a pure analytical standard, but there are other compounds detected in the non-targeted analysis that have yet to be validated. This study was the first to report FR compounds based on a direct injection method, coupled with a non-targeted screening workflow, at a trace level in a honey matrix. It also showed that a non-targeted workflow was effective to detect and identify unknown compounds present in the honey sample; hence, this provided a novel angle for the occurrence of FRs in air, with honey as a bio-indicator.

PMID: 37634584 [PubMed - as supplied by publisher]

Environ Sci Technicol. 2023 doi: 10.1021/acs.est.2c0939.
Authors: Xu Z, Tian L, Liu L, Goodyer CG, Hales BF, Bayen, S.

Abstract
To test the hypothesis that migration from the thermal labels on plastic film packaging is a major source of exposure to bisphenols and alternative color developers in food, we analyzed 140 packaging materials from packaged fresh food purchased in North America. No bisphenol A (BPA) was detected in either the packaging samples or thermal labels. However, significant amounts of bisphenol S (BPS) and alternative color developers (up to 214 μg/cm2) were present in thermal labels; their relative occurrence varied among stores. In a controlled experiment, we wrapped fish in film with a thermal label for 5 days at 4 °C. The fish in contact with the label contained BPS (≤1140 ng/g wet weight [ww]), 4-hydroxyphenyl 4-isoprooxyphenylsulfone (D-8) (≤230 ng/g ww), bis(2-chloroethyl)ether-4,4’-dihydroxydiphenyl sulfone monomer (D-90) (≤3.41 ng/g ww), and/or Pergafast-201 (≤1.87 ng/g ww). The corresponding film samples were then tested using migration cells for 10 days; significantly higher BPS migration was observed systematically from the films with thermal labels compared to plain films. This study provides evidence, for the first time, that BPS and alternative thermal label color developers migrate from packaging materials into food. Further, BPS migration significantly exceeded the European Union Specific Migration Limit (50 ng/g ww), suggesting that further risk assessment studies are warranted.

PMID: 36922386 [PubMed - as supplied by publisher]

Environ Int. 2023 171:107717 doi.org/10.1016/j.envint.2022.107717
Authors: Chi ZH, Goodyer CG, Hales BF, Bayen, S.

Abstract
Hundreds of xenobiotics, with very diverse origins, have been detected in human milk, including contaminants of emerging concern, personal care products and other current-use substances reflecting lifestyle. The routes of exposure to these chemicals include dermal absorption, ingestion and inhalation. Specific families of chemicals are dominant among human milk monitoring studies (e.g., organochlorine pesticides, bisphenol A, dioxins), even though other understudied families may be equally toxicologically relevant (e.g., food-processing chemicals, current-use plasticizers and flame retardants, mycotoxins). Importantly, the lack of reliable human milk monitoring data for some individual chemicals and, especially, for complex mixtures, is a major factor hindering risk assessment. Non-targeted screening can be used as an effective tool to identify unknown contaminants of concern in human milk. This approach, in combination with novel methods to conduct risk assessments on the chemical mixtures detected in human milk, will assist in elucidating exposures that may have adverse effects on the development of breastfeeding infants.

PMID: 36630790 [PubMed - as supplied by publisher]

Science of the Total Environment. 2022 840:156581.
Authors: Struzina L, Pineda Castro MA, Kubwabo C, Siddique S, Zhang G, Fan X, Tian L, Bayen S, Aneck-Hahn N, Bornman R, Chevrier J, Misunis M, Yargeau V.

Abstract
The occurrence of thirty-nine contaminants including plasticizers, bisphenols, and flame retardants in potable water from Montreal and South Africa was analyzed to determine their presence and concentrations in different water sources. In Montreal, five bottled water (BW) brands and three drinking water treatment plants (DWTP) were included. In South Africa, water was sampled from one urban DWTP located in Pretoria, Gauteng, and one rural DWTP located in Vhembe, along with water from the same rural DWTP which had been stored in small and large plastic containers. A combination of legacy compounds, typically with proven toxic effects, and replacement compounds was investigated. Bisphenols, Dechlorane-602, Dechlorane-603, and s-dechlorane plus (s-DP) were not detected in any water samples, and a-dechlorane plus (a-DP) was only detected in one sample from Pretoria at a concentration of 1.09 ng/L. Lower brominated polybrominated diphenyl ethers (PBDE)s were detected more frequently than higher brominated PBDEs, always at low concentrations of <2 ng/L, and total PBDE levels were statistically higher in South Africa than in Montreal. Replacement flame retardants, organophosphate esters (OPEs), were detected at statistically higher concentrations in Montreal's BW (68.56 ng/L), drinking water (DW) (421.45 ng/L) and Vhembe (198.33 ng/L) than legacy PBDEs. Total OPE concentrations did not demonstrate any geographical trend; however, levels were statistically higher in Montreal's DW than Montreal's BW. Plasticizers were frequently detected in all samples, with legacy compounds DEHP, DBP, and replacement DINCH being detected in 100 % of samples with average concentrations ranging from 6.89 ng/L for DEHP in Pretoria to 175.04 ng/L for DINCH in Montreal's DW. Total plasticizer concentrations were higher in Montreal than in South Africa. The replacement plasticizers (DINCH, DINP, DIDA, and DEHA) were detected at similar frequencies and concentrations as legacy plasticizers (DEHP, DEP, DBP, MEHP). For the compounds reported in earlier studies, the concentrations detected in the present study were similar to other locations. These compounds are not currently regulated in drinking water but their frequent detection, especially OPEs and plasticizers, and the presence of replacement compounds at similar or higher levels than their legacy compounds demonstrate the importance of further investigating the prevalence and the ecological or human health effects of these compounds.

PMID: 35697219 [PubMed - as supplied by publisher]

Talanta. 2022 253:123861. doi: 10.1016/j.talanta.2022.123861.
Authors: Xu Z, Chughtai H, Tian L, Liu L, Roy JF, Bayen S.

Abstract
Quantitative structure-retention relationship (QSRR) models can be used to predict the chromatographic retention time of chemicals and facilitate the identification of unknown compounds, notably with non-targeted analysis. In this study, QSRR models were developed from the data obtained for 178 pure chemical standards and four types of analytical columns (C18, phenylhexyl, pentafluorophenyl, cyano) in liquid chromatography quadrupole time-of-flight mass spectrometry (LC-Q-TOF-MS). First, different data partitioning ratios and feature selection methods [random forest (RF) and support vector machine (SVM)] were tested to build models to predict chromatographic retention times based on 2D molecular descriptors. The internal and external performances of the non-linear (RF) and corresponding linear predictive models were systematically compared, and RF models resulted in better predictive capacities [p &lt; 0.05, with an average PVE (proportion of variance explained) value of 0.89 ± 0.02] than linear models (0.79 ± 0.03). For each column, the resulting model was applied to identify leachables from actual plastic packaging samples. An in-depth investigation of the top 20 most intense molecular features revealed that all false-positives could be identified as outliers in the QSRR models (outside of the 95% prediction bands). Furthermore, analyzing a sample on multiple chromatographic columns and applying the associated QSRR models increased the capacity to filter false positives. Such an approach will contribute to a more effective identification of unknown or unexpected leachables in plastics (e.g. non-intended added substances), therefore refining our understanding of the chemical risks associated with food contact materials.

PMID: 36095943 [PubMed - as supplied by publisher]

Food Chemistry. 2022 385;132675.
Authors: Tian L, Zheng J, Pineda M, Yargeau V, Furlong D, Chevrier J, Bornman R, Obida V, Goodyer C, Bayen S.

Abstract
A sensitive method based on ultrasound-assisted liquid extraction coupled with liquid chromatography was applied to screen 18 plastic-related contaminants in 168 food composites (namely fish fillets, chicken breast, canned tuna, leafy vegetables, bread and butter) collected in Montreal (Canada), Pretoria and Vhembe (South Africa). Bisphenol A (BPA), bisphenol S (BPS) and seven plasticizers (di-n-butyl phthalate (DBP), diethyl phthalate (DEP), (2-ethylhexyl) phthalate (DEHP), di-(2-ethylhexyl) adipate (DEHA), di-isononyl phthalate (DINP), di-(isononyl)-cyclohexane-1,2-dicarboxylate (DINCH)) were detected in different foods from both countries. DBP and DEP were the most frequently detected contaminants in food collected in Montreal (75% for both) and DINP was the most frequently detected contaminant in food from South Africa (67%). DEHA concentration in packaged fish were significantly higher than the values for non-packaged fish (p < 0.01) suggesting that the packaging film can be one source of DEHA in fish.

PMID: 35305432 [PubMed - as supplied by publisher]

Canadian Journal of Law and Society. 2022 37(3) 1-19.
Authors: Ellis J.

Abstract
Building institutional and procedural bridges between science and policy is a vital role for law. Fundamental to the success of this work is the development of more sophisticated, nuanced understandings of scientific knowledge production than those which are current in legal and policy spheres. In this paper, I consider scientific controversies that have emerged in the field of human and environmental health impacts of endocrine disrupting chemicals (EDCs), notably around methodological approaches to identifying such chemicals and analysing the risks they pose. Building on literatures in the philosophy and sociology of science, I identify bases on which bridge-building between science and policy could proceed and discuss the role that legal normativity can play in those processes.

32 Duke Environmental Law and Policy Forum; Policy Forum. 2022 295-327.
Authors: Ellis J, Papaluca A, *Hamtiaux M, Hales BF, Robaire B.

Biol Reprod. 2022 106:613-627. doi: 10.1093/biolre/ioab216.
Authors: Rajkumar A, Luu T, Beal MA, Barton-Maclaren TS, Hales BF, Robaire B.

Abstract
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.

PMID: 34792101 [PubMed - as supplied by publisher]

Toxicol Sci. 2022 186:163-173. doi: 10.1093/toxsci/kfac001. Erratum for: Toxicol Sci. 2021 Nov 29.
Authors: Iskandarani L, McHattie T, Robaire B, Hales BF.

Erratum for
Effects of Bisphenols A, AF, and S on Endochondral Ossification and the Transcriptome of Murine Limb Buds.
Iskandarani L, McHattie T, Robaire B, Hales BF.
Toxicol Sci. 2022 May 26;187(2):234-253.

Abstract
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.

PMID: 34850234 [PubMed - as supplied by publisher]

Toxicol Sci. 2022 186(2):269-287. doi: 10.1093/toxsci/kfac012
Authors: Wang X, Luu T, Beal MA, Barton-Maclaren TS, Robaire B, Hales BF.

Abstract
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.

PMID: 35135005 [PubMed - as supplied by publisher]

Biol Reprod. 2022 107(3):858-868. doi: 10.1093/biolre/ioac10
Authors: Rajkumar A, Hales BF, Robaire B.

Abstract
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.

PMID: 35596243 [PubMed - as supplied by publisher]

Reprod Toxicol. 2021 102:10-23. doi: 10.1016/j.reprotox.2021.03.006.
Authors: Wang X, Hales BF, Robaire B.

Abstract
Flame retardants have been added to a variety of consumer products and are now found ubiquitously throughout the environment. Epidemiological, in vivo, and in vitro studies have shown that polybrominated diphenyl ether (PBDE) flame retardants may have a negative impact on human health; this has resulted in their phase-out and replacement by alternative flame retardants, such as hexabromocyclododecane (HBCDD), tetrabromobisphenol A (TBBPA), and organophosphate esters (OPEs). Evidence suggests that some of these chemicals induce ovarian dysfunction and thus may be detrimental to female fertility; however, the effects of many of these flame retardants on the ovary remain unclear. In this review, we present an overview of the effects of brominated and organophosphate ester flame retardants on ovarian function and discuss the possible mechanisms which may mediate these effects.

PMID: 33819575 [PubMed - as supplied by publisher]

Toxicol Sci. 2021 180(2):224-238. doi: 10.1093/toxsci/kfab012.
Authors: Rajkumar A, Luu T, Beal MA, Barton-Maclaren TS, Robaire B, Hales BF.

Abstract
Concerns about the potential adverse effects of bisphenol A (BPA) have led to an increase in the use of replacements, yet the toxicity data for several of these chemicals are limited. Using high-content imaging, we compared the effects of BPA, BPAF, BPF, BPS, BPM, and BPTMC in germ (C18-4 spermatogonial) and steroidogenic (MA-10 Leydig and KGN granulosa) cell lines. Effects on cell viability and phenotypic markers were analyzed to determine benchmark concentrations (BMCs) and estimate administered equivalent doses (AEDs). In all 3 cell lines, BPA was one of the least cytotoxic bisphenol compounds tested, whereas BPM and BPTMC were the most cytotoxic. Interestingly, BPF and BPS were cytotoxic only in MA-10 cells. Effects on phenotypic parameters, including mitochondria, lysosomes, lipid droplets, and oxidative stress, were both bisphenol- and cell-line specific. BPA exposure affected mitochondria (BMC: 1.2 μM; AED: 0.09 mg/kg/day) in C18-4 cells. Lysosome numbers were increased in MA-10 cells exposed to BPA or BPAF but decreased in KGN cells exposed to BPAF or BPM. Lipid droplets were decreased in C18-4 cells exposed to BPF and in MA-10 cells exposed to BPTMC but increased in BPF, BPM, and BPTMC-exposed KGN cells. BPA and BPM exposure induced oxidative stress in MA-10 and KGN cells, respectively. In summary, structurally similar bisphenols displayed clear cell-line-specific differences in BMC and AED values for effects on cell viability and phenotypic endpoints. This approach, together with additional data on human exposure, may aid in the selection and prioritization of responsible replacements for BPA.

PMID: 33501994 [PubMed - as supplied by publisher]

Crit Rev Food Sci Nutr. 2023;63(16):2687-2718. doi: 10.1080/10408398.2021.1980369.
Authors: Hales BF, Robaire B.

Abstract
Canning, as a preservation technique, is widely used to extend the shelf life as well as to maintain the quality of perishable foods. During the canning process, most of the microorganisms are killed, reducing their impact on food quality and safety. However, the presence of a range of undesirable chemical contaminants has been reported in canned foods and in relation to the canning process. The present review provides an overview of these chemical contaminants, including metals, polymeric contaminants and biogenic amine contaminants. They have various origins, including migration from the can materials, formation during the canning process, or contamination during steps required prior to canning (e.g. the disinfection step). Some other can-packaged foods (e.g. beverages or milk powder), which are not canned foods by definition, were also discussed in this review, as they have been frequently studied simultaneously with canned foods in terms of contamination. The occurrence of these contaminants, the analytical techniques involved, and the factors influencing the presence of these contaminants in canned food and can-packaged food are summarized and discussed.

PMID: 34583591 [PubMed - as supplied by publisher]

Andrology. 2020 8:915-923. doi: 10.1111/andr.12789.
Authors: Hales BF, Robaire B.

Abstract
Background: Environmental chemicals that interfere with the production and/or action of hormones may have adverse effects on male reproduction. This review focuses on the possible impact of exposure to flame retardant chemicals on male reproduction. Flame retardants are added to a wide variety of combustible materials to prevent fires from starting, slow their spread, and provide time to escape. However, these chemicals are often additive so they leach out into the environment. Governments have restricted the use of polybrominated diphenyl ether flame retardants based on evidence that they are persistent and bioaccumulate and have adverse effects on health. The phasing out of these "legacy" flame retardants has resulted in their replacement with alternatives, such as tetrabromobisphenol A and the organophosphate esters. Objective: To review the literature on the effects of brominated and organophosphate ester flame retardant chemicals on male reproduction. Methods: PubMed database was searched for studies reporting the effects of brominated and organophosphate ester flame retardants on male reproduction. Results: Cell-based, animal model, and human studies provide evidence that the polybrominated diphenyl ethers act as endocrine-disrupting chemicals; further, exposure during critical windows of development may be associated with a permanent impact on male reproduction. In vitro and animal model data are accumulating with respect to the effects of tetrabromobisphenol A and organophosphate esters, but few studies have evaluated their impact on human health.

PMID: 32216051 [PubMed - as supplied by publisher]

Toxicol Sci. 2020 Sep 25:kfaa145. doi: 10.1093/toxsci/kfaa145.
Authors: Yan H, Hales BF.

Abstract
Organophosphate esters have become widely used as flame retardants since the phase out of polybrominated diphenyl ethers. Previously, we demonstrated that some organophosphate esters, such as tert-butylphenyl diphenyl phosphate (BPDP), were more detrimental to endochondral ossification in murine limb bud cultures than one of the major polybrominated diphenyl ethers that they replaced, 2,2',4,4'-tetrabromodiphenyl ether. Here, we used a transcriptomic approach to elucidate the mechanism of action of BPDP in the developing limb. Limb buds collected from gestation day 13 CD1 mouse embryos were cultured for 3 or 24 h in the presence of vehicle, 1 μM, or 10 μM BPDP. RNA sequencing analyses revealed that exposure to 1 µM BPDP for 24 h increased the expression of 5 transcripts, including Ihh, and decreased 14 others, including Gli1, Ptch1, Ptch2, and other targets of Hedgehog (Hh) signaling. Pathway analysis predicted the inhibition of Hh signaling. Attenuation of Hh signaling activity began earlier and reached a greater magnitude after exposure to 10 µM BPDP. Because this pathway is part of the regulatory network governing endochondral ossification, we used a known Hh agonist, purmorphamine, to determine the contribution of Hh signaling inhibition to the negative impact of BPDP on endochondral ossification. Cotreatment of limbs with purmorphamine rescued the detrimental morphological changes in the cartilage template induced by BPDP exposure though it did not restore the expression of key transcription factors, Runx2 and Sp7, to control levels. These data highlight Hh signaling as a developmentally important pathway vulnerable to environmental chemical exposures.

PMID: 32976586 [PubMed - as supplied by publisher]

Food Chemistry. 2020 316: 126942.
Authors: Tian L, Zheng J, Goodyer C, Bayen S.

Abstract
A non-targeted screening method based on ultrasound-assisted extraction followed by high-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (HPLC-QTOF-MS) was developed to screen for the presence of plastic-related chemicals (PRCs) in different types of food (fish, chicken, canned tuna, leafy vegetables, bread and butter). Eleven bisphenols were used as targeted compounds. Instrument linearity (r2 ≥0.98), inter-day precision (RSD ≤9.0%) as well as method detection limits (MDLs below 3.6 ng g-1) were satisfactory. Recoveries of the eleven bisphenols ranged from 76% to 122% among the different food matrices. The method was applied to food collected from Montreal, Canada in 2017-2018. The non-targeted screening approach identified a range of contaminants in different food matrices, including BPA, BPS, bis(2-ethylhexyl) adipate, dibutyl adipate, hexadecyl methacrylate and Irganox®1076. Further research is suggested to investigate the concentration of these PRCs, the consumption habits of average and specific populations and the potential routes of contamination.

PMID: 32407999 [PubMed - as supplied by publisher]

Food Chem. 2020 328:126999.
Authors: Tian L, Goodyer CG, Zheng J, Bayen S.

Abstract
The thermal degradation of bisphenol A (BPA) and bisphenol S (BPS) was investigated in water and fish (cod, basa) fillets. Ultrasound assisted solvent extraction followed by high performance liquid chromatography coupled with quadrupole time of flight mass spectrometry (HPLC-QTOF-MS) was used to analyze residues in fish. Good instrumental linearity (r2 > 0.99) and recoveries (83.3-128.4%) were achieved. BPA and BPS did not degrade (1 h; 100 °C) in water (<0.1% degradation) but degraded in fish matrices. The degradation percentage of BPA was 33.0 ± 1.5% and 35.4 ± 1.2% in incurred and spiked cod, respectively; and the degradation percentage of BPS was 34.7 ± 1.7% and 37.5 ± 1.4% in incurred and spiked basa, respectively. The degradation products in spiked samples were different from those in the incurred group under the same conditions. This first study on the thermal degradation of plastic-related chemicals in food using a non-targeted approach will contribute to the refining of food safety risk assessments./p>

PMID: 32474236 [PubMed - as supplied by publisher]

Toxicological Sciences. 2019 168:420-429.
Authors: Yan H, Hales BF.

Abstract
Phasing out the usage of polybrominated diphenyl ether (PBDE) flame retardants (FRs) in consumer products led to their widespread replacement with organophosphate ester (OPE) FRs, despite scarce safety data. PBDE exposures were associated with the suppression of endochondral ossification but little is known about the effects of OPEs on bones. Here, we used a novel ex vivo murine limb bud culture system to compare the effects of 2,2',4,4'-tetrabromodiphenyl ether (BDE-47) with those of several OPEs. Gestation day 13 embryos were collected from transgenic CD1 mice expressing fluorescent markers for the major stages of endochondral ossification: COL2A1-ECFP (chondrogenesis), COL10A1-mCherry (early osteogenesis), and COL1A1-YFP (late osteogenesis). Limbs were excised and cultured for 6 days in the presence of vehicle, BDE-47, or an OPE FR: triphenyl phosphate (TPHP), tert-butylphenyl diphenyl phosphate (BPDP), tris(methylphenyl) phosphate (TMPP), or isopropylated triphenyl phosphate (IPPP). BDE-47 (50 μM) decreased the extent of chondrogenesis in the digits and COL1A1-YFP expression in the radius and ulna relative to control. In comparison, concentrations of ≥1 μM of all 4 OPEs limited chondrogenesis; osteogenesis (both COL10A1-mCherry and COL1A1-YFP fluorescence) was markedly inhibited at concentrations ≥3 μM. The expression of Sox9, the master regulator of chondrogenesis, was altered by BDE-47, TPHP, and BPDP. BDE-47 exposure had minimal impact on the expression of Runx2 and Sp7, which drive osteogenesis, whereas TPHP and BPDP both suppressed the expression of these transcription factors. These data suggest that OPE FRs may be more detrimental to bone formation than their brominated predecessors.

PMID: 30561715 [PubMed - as supplied by publisher]

Academy of Management Journal, Published Online: 2 May 2019,
Authors: Hardy C, Maguire S.

Abstract
The contemporary ‘risk society’ is associated with the emergence of a wide range of risks characterised by uncertainty and unfamiliarity. These ‘novel’ risks pose a major challenge for organizations: their negative effects may be significant, but prevailing risk assessment techniques are limited in their ability to identify them. Building on our prior work on the chemical bisphenol A (BPA), this study examines how organizations deal with novel risks. It finds that organizations engage in ‘risk translation’ by translating equivocality associated with the novel risk into more familiar risks, which provide them with a clearer basis and guide for action. As organizations take actions to manage these translated risks, an ‘ecology of risks’ evolves which, over time, allows for the construction of a novel risk. The study contributes to research on organizing and risk by theorizing how organizations respond to novel risks, as well as by highlighting the role of translated organizational risks in constructing novel risks and shaping societal responses to grand challenges.

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