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.

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.

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 expressionbased BMD. At 12.0 mg/kg, there were 38, 33, and 65 DEG (P value <.005; fold change >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-adverseeffect-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 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.

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.

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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.

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