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Bisphenol A (BPA) is a chemical used in the manufacturing of plastics to which human exposure is ubiquitous. Numerous studies have linked BPA exposure to many adverse health outcomes prompting the replacement of BPA with various analogues including bisphenol-F (BPF) and bisphenol S (BPS). Other bisphenols are used in various consumer applications, such as 3,3′,5,5’-Tetrabromobisphenol A (TBBPA), which is used as a flame retardant. Few studies to date have examined the effects of BPA and its analogues in stem cells to explore potential developmental impacts. Here we used transcriptomics to investigate similarities and differences of BPA and three of its analogues in the estrogen receptor negative, human embryonic stem cell line H9 (WA09). H9 cells were exposed to increasing concentrations of the bisphenols and analyzed using RNA-sequencing. Our data indicate that BPA, BPF, and BPS have similar potencies in inducing transcriptional changes and perturb many of the same pathways. TBBPA, the least structurally similar bisphenol of the group, exhibited much lower potency. All bisphenols robustly impacted gene expression in these cells, albeit at concentrations well above those observed in estrogen-positive cells. Overall, we provide a foundational data set against which to explore the transcriptional similarities of other bisphenols in embryonic stem cells, which may be used to assess the suitability of chemical grouping for read-across and for preliminary potency evaluation.

Acrylamide (AA) exposure causes increased incidence of forestomach, lung, and Harderian gland tumors in male mice. One hypothesized mode of action (MOA) for AA-carcinogenicity includes genotoxicity/mutagenicity as a key event, possibly resulting from AA metabolism to the direct genotoxic metabolite glycidamide. Alternatively, altered calcium signaling (CS) has been proposed as a central key event in the MOA. To examine the plausibility of these proposed MOAs, RNA-sequencing was performed on tumor target tissues: Harderian glands (the most sensitive tumor target tissue in the rodent 2-year cancer bioassay) and lungs of AA-exposed male CD-1 mice. Animals were exposed to 0.0, 1.5, 3.0, 6.0, 12.0, or 24.0 mg AA/kg bw-day in drinking water for 5, 15, or 31 days. We observed a pronounced effect on genes involved in CS and cytoskeletal processes in both tissues, but no evidence supporting a genotoxic MOA. Benchmark dose modeling suggests transcriptional points of departure (PODs) of 0.54 and 2.21 mg/kg bw-day for the Harderian glands and lungs, respectively. These are concordant with PODs of 0.17 and 1.27 mg/kg bw-day derived from the cancer bioassay data for these tissues in male mice, respectively. Overall, this study supports the involvement of CS in AA-induced mouse carcinogenicity, which is consistent with a recently proposed CS-based MOA in rat thyroid, and with other published reports of aberrant CS in malignant tumors in rodents and humans.

Graphical abstract, Highlights • Pentabrominated diphenyl ether (PBDE) mixture was a multispecies carcinogen causing liver tumors in male and female rats and mice. • Hras or Ctnnb1 mutations characterized the PBDE-induced liver tumors. • PBDE-induced liver tumors increased with increasing PBDE exposure., Pentabrominated diphenyl ether (PBDE) flame retardants have been phased out in Europe and in the United States, but these lipid soluble chemicals persist in the environment and are found human and animal tissues. PBDEs have limited genotoxic activity. However, in a 2-year cancer study of a PBDE mixture (DE-71) (0, 3, 15, or 50 mg/kg (rats); 0, 3, 30, or 100 mg/kg (mice)) there were treatment-related liver tumors in male and female Wistar Han rats [Crl:WI(Han) after in utero/postnatal/adult exposure, and in male and female B6C3F1 mice, after adult exposure. In addition, there was evidence for a treatment-related carcinogenic effect in the thyroid and pituitary gland tumor in male rats, and in the uterus (stromal polyps/stromal sarcomas) in female rats. The treatment-related liver tumors in female rats were unrelated to the AhR genotype status, and occurred in animals with wild, mutant, or heterozygous Ah receptor. The liver tumors in rats and mice had treatment-related Hras and Ctnnb mutations, respectively. The PBDE carcinogenic activity could be related to oxidative damage, disruption of hormone homeostasis, and molecular and epigenetic changes in target tissue. Further work is needed to compare the PBDE toxic effects in rodents and humans.

Background: The complex and dynamic nature of the tumor-immune microenvironment presents challenges for identification of robust and predictive biomarkers in immuno-oncology (IO). Multiplex immunohistochemistry (mIHC) facilitates the ability to detect, phenotype, and quantify spatial relationships of cells within the tumor microenvironment (TME). Gene expression profiling allows for sensitive and high-throughput analysis of genes and signatures associated with the tumor, the immune response, and the TME, allowing examination of tumor-immune cell interactions. We used these approaches to generate multiple data sets from a cohort of HNSCC tumors and evaluated the correlation of the various analyte detection methods. These complementary technologies provide useful tools in the IO biomarker toolkit. Methods: Formalin-fixed paraffin-embedded (FFPE) specimens from HNSCC patients were cut into 5µm sections for all technologies. Multiplex fluorescent IHC was performed for TME markers CD3, CD8, PD-L1, PD1, CD68, Granzyme B, Ki67, and panCK/SOX10. Visualization and data analysis were performed with an Akoya Vectra Polaris and Akoya inForm and HALO software (Indica Labs). Data analysis included cell identification, phenotyping, spatial relationships, and quantitative digital pathology. Gene expression for 770 genes was performed utilizing the NanoString PanCancer IO 360 Gene Expression Panel. Transcripts were quantitated using a NanoString nCounter and target gene counts were normalized to internal housekeeping genes. Results: Analysis of the multiplex fluorescent IHC indicated a range of expression for the assayed TME biomarkers for the different tumor samples. Quantitative analysis of mIHC phenotype counts and normalized RNA counts for the targets contained in the antibody panel revealed a significant correlation between the analytic methods. Comparison of mIHC phenotype counts with a previously validated IO gene signature containing a broader set of IO-relevant genes, the Tumor Information Signature, (Ayers, et al., J Clin Invest. 2017; 127:2930) also showed a correlation. Conclusions: The technologies described above enable the investigation of the TME for use in biomarker discovery, drug discovery, and IO pathway interrogation. These technologies can be used in parallel to uncover roles in the biomarker discovery pathway for genes of interest. The combination of mIHC and gene expression panels can be used to screen a broad range of targets, that then can be further investigated using the spatial analysis capabilities of mIHC to gain greater insight into the immune infiltrate density at any singular area of the TME. Citation Format: Carlee Hemphill, Timothy Maynor, John Bauman, Caitlin Schroyer, Jeffery Shuster, Thomas Turi, Steven M. Anderson. Multiplex immunohistochemistry, spatial analysis, and gene expression profiling of the tumor-immune microenvironment in HNSCC tumors [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2637.

Background: The degree of methylation in the promoter region of the O-6-methylguanine-DNA methyltransferase (MGMT) gene is considered a prognostic biomarker of patient outcomes in Glioblastoma multiforme (GBM). There are a number of different assays currently available for the determination of MGMT gene methylation status, with the assays using different methods and evaluating different CpG sites in the MGMT promoter and in exon 1 to determine methylation status. There is the possibility that these methods may produce variant methylation results and thus lead to different patient treatment decisions based on the test results. Methods: We have provided MGMT testing services for clinical samples based upon a published assay (Kam-Morgan et al. 2009 AMP Annual Meeting; Hegi et al., 2019, Clin Cancer Res. 25:1809) for more than a decade. Because of the need to change instrumentation we have further optimized the methodology and analytically validated an MGMT methylation assay for possible use as an in vitro diagnostic device. Formalin-fixed-paraffin-embedded (FFPE) specimens from GBM patients were evaluated using standard pathology methods: the tumor region is identified, and the corresponding tumor region from unstained slides macro-dissected. DNA was isolated by standard methods and analyzed by methylation specific real-time PCR. The copy number of methylated MGMT based on 8 CpG sites was determined and normalized to the copy number of β-Actin gene (ACTB). The following parameters were evaluated: linearity, precision, and concordance with other testing methods. Results: The assay was shown to demonstrate linearity across input DNA ranges of 2.75 ng to 2500 ng DNA isolated from FFPE clinical samples. Assay precision was measured in clinical specimens with multiple replicates, instruments, and operators, over 6 non-consecutive days of testing. Methylation status for each sample was consistent throughout the study and across all variables. Intra-tumor heterogeneity was assessed in serial sections from clinical samples over a distance of approximately 150 µm of tumor. Results: (methylated or un-methylated) were concordant for >99% of assays. An orthogonal method of determining methylation at the 8 CpG sites was performed by pyrosequencing. Results indicated good correlation of the two methods with 90% agreement in calls between the methods. Conclusions: The assay described here provides a robust and reproducible measure of MGMT status in GBM tissue samples. Citation Format: Benjamin Avery, Neal Englert, Celso Espinoza, Timothy Maynor, Christopher Phillips, Jim Yan, Thomas Turi, Jeffery Shuster, Steven M. Anderson. Development of a robust MGMT promoter methylation assay for glioblastoma multiforme [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 151.

Acrylamide (AA) at high exposure levels is neurotoxic, induces testicular toxicity, and increases dominant lethal mutations in rats. RNA-sequencing in testes was used to identify differentially expressed genes (DEG), explore AA-induced pathway perturbations that could contribute to AA-induced testicular toxicity and then used to derive a benchmark dose (BMD). Male F344/DuCrl rats were administered 0.0, 0.5, 1.5, 3.0, 6.0, or 12.0 mg AA/kg bw/d in drinking water for 5, 15, or 31 days. The experimental design used exposure levels that spanned and exceeded the exposure levels used in the rat dominant lethal, 2-generation reproductive toxicology, and cancer bioassays. The time of sample collection was based on previous studies that developed gene expression–based BMD. At 12.0 mg/kg, there were 38, 33, and 65 DEG ( P value 1.5) in the testes after 5, 15, or 31 days of exposure, respectively. At 31 days, there was a dose-dependent increase in the number of DEG, and at 12.0 mg/kg/d the top three functional clusters affected by AA exposure were actin filament organization, response to calcium ion, and regulation of cell proliferation. The BMD lower 95% confidence limit using DEG ranged from 1.8 to 6.8 mg/kg compared to a no-observed-adverse-effect-level of 2.0 mg/kg/d for male reproductive toxicity. These results are consistent with the known effects of AA on calcium signaling and cytoskeletal actin filaments leading to neurotoxicity and suggest that AA can cause rat dominant lethal mutations by these same mechanisms leading to impaired chromosome segregation during cell division.

Acrylamide (AA) exposure in 2-year cancer bioassays leads to thyroid, but not liver, adenomas and adenocarcinomas in rats. Hypothesized modes of action (MOAs) include genotoxicity/mutagenicity, or thyroid hormone dysregulation. To examine the plausibility of these two or any alternative MOAs, RNA-sequencing was performed on the thyroids and livers of AA-exposed rats, in parallel with measurement of genotoxicity (blood micronucleus and Pig-a mutant frequency) and serum thyroid hormone levels, following the exposure of male Fischer 344/DuCrl rats to 0.0, 0.5, 1.5, 3.0, 6.0, or 12.0 mg AA/kg bw-day in drinking water for 5, 15, or 31 days. Differentially expressed genes in both tissues provided marginal support for hormonal and genotoxic MOAs, which was consistent with negative/equivocal genotoxicity assay and marginal changes in thyroid hormone levels. Instead, there was a pronounced effect on calcium signaling/cytoskeletal genes in the thyroid. Benchmark dose modeling of RNA-sequencing data for the calcium signaling pathway suggests a point of departure (POD) of 0.68 mg/kg bw-day, which is consistent with a POD of 0.82 mg/kg bw-day derived from the thyroid 2-year cancer bioassay data. Overall, this study suggests a novel MOA for AA-induced thyroid carcinogenicity in male rats centered around perturbation of calcium signaling.

The use of brominated flame retardants and incineration of bromine-containing materials has lead to an increase in polybrominated dibenzo-p-dioxins and dibenzofurans (PBDD/Fs) in the environment. Measurable amounts of PBDD/Fs have been detected in soil, seafood, and human breast milk and serum. Studies indicate that the relative potencies of some PBDD/Fs based on enzyme induction are equivalent to those of some polychlorinated dibenzo-p-dioxins and dibenzofurans. To assess the humoral immunity relative potencies of PBDD/Fs and compare them to their chlorinated analogs, female B6C3F1/N mice received a single oral exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), 2,3,7,8-tetrabromodibenzofuran (TBDF), 2,3,7,8-tetrachlorodibenzofuran (TCDF), 1,2,3,7,8-pentabromodibenzofuran (1PeBDF), 1,2,3,7,8-pentachlorodibenzofuran (1PeCDF), 2,3,4,7,8-pentabromodibenzofuran (4PeBDF), 2,3,4,7,8-pentachlorodibenzofuran (4PeCDF), 2,3-dibromo-7,8-dichlorodibenzo-p-dioxin (DBDCDD), or 2,3,7-tribromodibenzo-p-dioxin (TriBDD). Inhibition of the immunoglobulin M (IgM) antibody forming cell response was measured 4 days following immunization with sheep red blood cells. The data were fit to a Hill model to estimate the ED50 for inhibition. Expression of xenobiotic metabolizing enzyme (XME) and thyroxine transport protein (Ttr) genes in liver was measured by PCR to assess aryl hydrocarbon-mediated responses. TCDD, TBDF, TCDF, 1PeBDF, 4PeBDF, 4PeCDF, and DBDCDD suppressed the IgM antibody response and Ttr gene expression, and upregulated phase I XME genes. 1PeCDF suppressed the IgM antibody response but only upregulated phase I XME genes; TriBDD had no effect on antibody response. The rank order of potency (ED50) for these chemicals was TCDD>TBDF>4PeBDF>TCDF/4PeCDF/1PeBDF>1PeCDF. Whereas TCDD was the most potent compound tested, the brominated analogs were more potent than their chlorinated analogs, suggesting that these compounds should be considered in toxic equivalency factor evaluation and risk assessment.

The mammalian genome is transcribed into mRNAs that code for protein and a broad spectrum of other noncoding (nc) RNA products. Long ncRNAs (lncRNA), defined as ncRNA species > 200 nucleotides long, are emerging as important epigenetic regulators of gene expression that are involved in a spectrum of biological processes of relevance to toxicology. We conducted a gene expression profiling study in the livers of female B6C3F1 mice exposed to the carcinogen furan at 0.0, 1.0, and 2.0mg/kg (noncarcinogenic doses) and at 4.0 and 8.0mg/kg (carcinogenic doses) for 3 weeks. LncRNA differential expression showed a nonlinear dose response with none differentially expressed at 1.0 or 2.0mg/kg, 2 lncRNAs at 4.0mg/kg furan, and 83 at 8mg/kg, representing 13.3% (83/632) of the total number of differentially expressed transcripts. Among the lncRNAs observed, two lncRNAs examined showed transcriptional clustering with nearby protein-coding genes. LincRNA-p21 is an antisense transcript that is 15kb downstream from Cdkn1a locus and appears to be cotranscribed with the protein coding gene Cdkn1a at 8.0mg/kg furan. In a separate independent study, RNA samples from the livers of mice administered benzo(a)pyrene also demonstrated increased levels of Cdkn1a and the antisense lincRNA-p21 transcript. These data demonstrate that lncRNAs are transcriptional targets of furan exposures associated with levels of furan that are cytotoxic and induce cell proliferation. In addition, certain lncRNA transcripts are associated with the expression of nearby coding protein genes. We hypothesize that lncRNAs have potential as epigenetic biomarkers of carcinogenic exposures.

e16036 Background: Prostate cancer is the most common cause of cancer death in men over age 75, with most cases progressing to castration resistant prostate cancer (CRPC). Docetaxel remains the sta...