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Anaplastic thyroid carcinoma is arguably the most lethal human malignancy. It often co-occurs with differentiated thyroid cancers, yet the molecular origins of its aggressivity are unknown. We sequenced tumor DNA from 329 regions of thyroid cancer, including 213 from patients with primary anaplastic thyroid carcinomas. We also whole genome sequenced 9 patients using multi-region sequencing of both differentiated and anaplastic thyroid cancer components. Using these data, we demonstrate thatanaplastic thyroid carcinomas have a higher burden of mutations than other thyroid cancers, with distinct mutational signatures and molecular subtypes. Further, different cancer driver genes are mutated in anaplastic and differentiated thyroid carcinomas, even those arising in a single patient. Finally, we unambiguously demonstrate that anaplastic thyroid carcinomas share a genomic origin with co-occurring differentiated carcinomas and emerge from a common malignant field through acquisition of characteristic clonal driver mutations., Competing Interests: Declaration of interests All authors declare that they have no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

People with Li-Fraumeni syndrome (LFS) harbor a germline pathogenic variant in the TP53 tumor suppressor gene, face a near 100% lifetime risk of cancer, and routinely undergo intensive surveillance protocols. Liquid biopsy has become an attractive tool for a range of clinical applications, including early cancer detection. Here, we provide a proof-of-principle for a multimodal liquid biopsy assay that integrates a targeted gene panel, shallow whole-genome, and cell-free methylated DNA immunoprecipitation sequencing for the early detection of cancer in a longitudinal cohort of 89 LFS patients. Multimodal analysis increased our detection rate in patients with an active cancer diagnosis over uni-modal analysis and was able to detect cancer-associated signal(s) in carriers prior to diagnosis with conventional screening (positive predictive value = 67.6%, negative predictive value = 96.5%). Although adoption of liquid biopsy into current surveillance will require further clinical validation, this study provides a framework for individuals with LFS., Significance: By utilizing an integrated cell-free DNA approach, liquid biopsy shows earlier detection of cancer in patients with LFS compared with current clinical surveillance methods such as imaging. Liquid biopsy provides improved accessibility and sensitivity, complementing current clinical surveillance methods to provide better care for these patients. See related commentary by Latham et al., p. 23. This article is featured in Selected Articles from This Issue, p. 5., (©2023 The Authors; Published by the American Association for Cancer Research.)

Objectives: Although human papillomavirus positive (HPV+) oropharyngeal squamous cell carcinoma (OPSCC) patients typically experience excellent survival, 15-20 % of patients recur after treatment with chemotherapy and radiation. Therefore, there is a need for biomarkers of treatment failure to guide treatment intensity., Materials and Methods: Whole genome sequencing was carried out on HPV+OPSCC patients who were primarily treated with concurrent chemotherapy (cisplatin) and radiation. We then explored whether the loss of LRP1Bwas sufficient to drive an aggressive phenotype, and promote a resistance to cisplatin and radiation therapy both in vitro using HPV+ cell lines (93VU147T, UMSCC47, UWO37 and UWO23) and in vivo., Results: Through integrative genomic analysis of three HPV+OPSCC tumour datasets, we identified that deletion of LRP1B was enriched in samples that recurred following chemo-radiation. Knockdown using siRNA in four HPV+ cell lines (UWO23, UWO37, UMSCC47 and 93VU147T) resulted in increased proliferation of all cases. CRISPR/Cas9 deletion of LRP1B in the same cell line panel demonstrated increased proliferation, clonogenic growth and migration, as well as resistance to both cisplatin and radiation in LRP1B deleted cells compared to their respective non-targeting control cells. Cell line derived xenograft studies indicated that the LRP1B knockout tumours were more resistant to cisplatin and radiation therapy compared to their controls invivo., Conclusion: Taken together, our work implicates LRP1B deletion as a potential biomarker for identifying treatment resistant HPV+ OPSCC cases., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier Ltd.)

Purpose: To evaluate the use of blood cell-free DNA (cfDNA) to identify emerging mechanisms of resistance to PARP inhibitors (PARPi) in high-grade serous ovarian cancer (HGSOC)., Experimental Design: We used targeted sequencing (TS) to analyze 78 longitudinal cfDNA samples collected from 30 patients with HGSOC enrolled in a phase II clinical trial evaluating cediranib (VEGF inhibitor) plus olaparib (PARPi) after progression on PARPi alone. cfDNA was collected at baseline, before treatment cycle 2, and at end of treatment. These were compared with whole-exome sequencing (WES) of baseline tumor tissues., Results: At baseline (time of initial PARPi progression), cfDNA tumor fractions were 0.2% to 67% (median, 3.25%), and patients with high ctDNA levels (>15%) had a higher tumor burden (sum of target lesions; P = 0.043). Across all timepoints, cfDNA detected 74.4% of mutations known from prior tumor WES, including three of five expected BRCA1/2 reversion mutations. In addition, cfDNA identified 10 novel mutations not detected by WES, including seven TP53 mutations annotated as pathogenic by ClinVar. cfDNA fragmentation analysis attributed five of these novel TP53 mutations to clonal hematopoiesis of indeterminate potential (CHIP). At baseline, samples with significant differences in mutant fragment size distribution had shorter time to progression (P = 0.001)., Conclusions: Longitudinal testing of cfDNA by TS provides a noninvasive tool for detection of tumor-derived mutations and mechanisms of PARPi resistance that may aid in directing patients to appropriate therapeutic strategies. With cfDNA fragmentation analyses, CHIP was identified in several patients and warrants further investigation., (©2023 The Authors; Published by the American Association for Cancer Research.)

Objective: We investigated the autoantibody (autoAb) profiles in ANA+ individuals lacking systemic autoimmune rheumatic disease (SARD) and early SARD patients to determine the key differences between these groups and identify factors that are associated with an increased risk of symptomatic progression within the next 2 years in ANA+ individuals., Methods: Using custom antigen (Ag) microarrays, 144 IgM and IgG autoAbs were surveyed in 84 asymptomatic and 123 symptomatic (48 UCTD and 75 SARD patients) ANA+ individuals. AutoAbs were compared in ANA+ individuals lacking a SARD diagnosis with ≥2 years follow-up (n = 52), including all those who demonstrated progression (n = 14) during this period, with changes over time assessed in a representative subset., Results: We show that ANA+ individuals have autoAb to many self-Ags that are not being captured by current screening techniques and very high levels of these autoAbs are predominantly restricted to early SARD patients, with SLE patients displaying reactivity to many more autoAgs than the other groups. In general, the symptoms that developed in progressors mirrored those seen in SARD patients with similar patterns of autoAbs. Only anti-Ro52 Abs were found to predict progression (positive predictive value 46%, negative predictive value 89%). Surprisingly, over 2 years of follow-up the levels of autoAbs remained remarkably stable regardless of whether individuals progressed or not., Conclusion: Our findings strongly argue that development of assays with an expanded set of auto-Ags and enhanced dynamic range would improve the diagnostic and prognostic ability of autoAb testing., (© The Author(s) 2021. Published by Oxford University Press on behalf of the British Society for Rheumatology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Anaplastic thyroid cancer (ATC) is a rare, but nearly uniformly fatal disease that is typically resistant to chemotherapy and radiation. Alternative strategies to target this cancer at a molecular level are necessary in order to improve dismal outcomes for ATC patients. We examined the effects of flavopiridol, a CDK inhibitor, in a panel of ATC cell lines. When cell lines were treated over a ten-point concentration range, CAL62, KMH2 and BHT-101 cell lines had a sub micromolar half-maximal inhibitory concentration, while no effect was seen in the non-cancerous cell line IMR-90. Flavopiridol treatment resulted in decreased levels of the cell cycle proteins CDK9 and MCL1, and induced cell cycle arrest. Flavopiridol also decreased the in vitro ability of ATC cells to form colonies and impeded migration using a transwell migration assay. In vivo, flavopiridol decreased tumor weight and tumor volume over time in a patient-derived xenograft model of ATC. Given the observed in vitro and in vivo activity, flavopiridol warrants further investigation for treatment of ATC., Competing Interests: The authors have declared that no competing interests exist.

Sex differences have been observed in multiple facets of cancer epidemiology, treatment and biology, and in most cancers outside the sex organs. Efforts to link these clinical differences to specific molecular features have focused on somatic mutations within the coding regions of the genome. Here we report a pan-cancer analysis of sex differences in whole genomes of 1983 tumours of 28 subtypes as part of the ICGC/TCGA Pan-Cancer Analysis of Whole Genomes (PCAWG) Consortium. We both confirm the results of exome studies, and also uncover previously undescribed sex differences. These include sex-biases in coding and non-coding cancer drivers, mutation prevalence and strikingly, in mutational signatures related to underlying mutational processes. These results underline the pervasiveness of molecular sex differences and strengthen the call for increased consideration of sex in molecular cancer research.

Objectives: Survival in head and neck squamous cell carcinoma (HNSCC) has been associated with patient sex, typically with males experiencing poorer outcomes. It is unclear if this disparity is based in divergent tumor biology. We analyzed the TCGA HNSCC cohort to uncover disparities in the somatic single nucleotide variation (SNV), copy number alteration (CNA) and mRNA abundance profiles between males and females. Critically, we stratified our results by tumor HPV status to control for this significant confounder., Methods: SNV, CNA and mRNA abundance differences between males and females were compared separately for the HPV-positive (n = 67) and negative (n = 431) TCGA HNSCC cohorts. Overall survival outcomes were compared in males and females in both HPV-positive and HPV-negative subsets of patients., Results: Females were found to have poorer overall survival than males (p = 0.048), largely due to higher rates of HPV-positive disease among men. SNV analysis revealed that in HPV-positive disease, there were no differences by sex after accounting for the false discovery rate (FDR). In HPV-negative tumors, BRWD3 mutations occurred more frequently in the tumors of female patients compared to males after adjusting for the FDR (p = 0.02). Further, HPV-negative BRWD3 mutant tumors were found to have significantly worse 5-year overall survival compared to wildtype on multivariate analysis (p = 0.02). There were 88 heterozygous deletions and 14 amplifications that were differentially altered between male and female HPV-negative tumors and associated with expression changes. Pathway analysis of these genes revealed that tumors from males were enriched in five pathways including chemokine and phosphophatidylinositol signaling., Conclusions: Reanalysis of the TCGA HNSCC dataset stratified by sex revealed that males in this cohort had a significant survival advantage, due to a higher proportion of HPV-positive disease. Mutations in BRWD3 were more frequent in HPV-negative tumors of females and were associated with poorer overall survival. BRWD3 may represent a novel biomarker of patient outcomes, but will require additional validation., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

In rats, direct exposure to TCDD causes myriad toxicities. Exposed rats experience hepatotoxicity, wasting syndrome and immune suppression, amongst others. "Inherited exposure", as occurs in the F3 generation of directly exposed F0 animals, has also been shown to cause toxicity: both male and female F3 rats demonstrate an increased incidence of adult onset disease, females also display reproductive abnormalities and increased incidence of ovarian diseases while males show increased incidence of kidney disease and an altered sperm epigenome. Here, we explore the hepatic transcriptomic profile of male and female F3 Sprague-Dawley rats bred through the paternal germ line from F0 dams exposed to a single dose of TCDD (0, 30, 100, 300 or 1000 ng/kg body weight) by oral gavage. We hypothesize that RNA transcripts with altered abundance in livers of unexposed F3 progeny of treated F0 Sprague-Dawley rats may result from epigenetic modifications to the genome. We further survey patterns of differential methylation within male F3 rat testis. Female F3 rats demonstrated more TCDD-mediated hepatic transcriptomic changes than males, with differences primarily in the lowest dose group. In testis from male F3 rats, multiple olfactory receptors displayed patterns of differential methylation. Hypermethylation of Egfr and Mc5r among testes from TCDD lineage rats was observed, but without corresponding changes in hepatic mRNA abundance. Further studies examining these differences in other tissue types are warranted.

Background: The genomic landscape of head and neck cancer has been reported through The Cancer Genome Atlas project. We attempt to determine if high-risk human papillomavirus (HPV) or frequently mutated genes are correlated with survival in an oral cancer cohort., Methods: Patient demographic data along with data from final pathology was collected. Tumor DNA was analyzed using a custom Illumina targeted sequencing panel. Five high-risk HPV types were tested by qPCR. Statistical analyses were used to identify associations between patient outcome and mutational status., Results: High-risk HPV types were identified in 7% of cases; HPV status was not associated with survival. Mutations were identified in TP53, TERT promoter, & PIK3CA. Mutations in TP53 were significantly associated with poorer overall survival on multi-variate analysis ( p  = 0.03)., Conclusions: Mutations in TP53 were associated with poor patient survival. Expanding our sample size may identify further predictors of outcome to direct customized cancer care., Competing Interests: Competing interestsThe authors declare that they have no competing interests., (© The Author(s) 2019.)

The aryl hydrocarbon receptor (AHR) mediates many toxic effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). However, the AHR alone does not explain the widely different outcomes among organisms. To identify the other factors involved, we evaluated three transgenic mouse lines, each expressing a different rat AHR isoform (rWT, DEL, and INS) providing widely different resistance to TCDD toxicity, as well as C57BL/6 and DBA/2 mice which exhibit a ~ tenfold divergence in TCDD sensitivity (exposures of 5-1000 μg/kg TCDD). We supplement these with whole-genome sequencing, together with transcriptomic and proteomic analyses of the corresponding rat models, Long-Evans (L-E) and Han/Wistar (H/W) rats (having a ~ 1000-fold difference in their TCDD sensitivities; 100 μg/kg TCDD), to identify genes associated with TCDD-response phenotypes. Overall, we identified up to 50% of genes with altered mRNA abundance following TCDD exposure are associated with a single AHR isoform (33.8%, 11.7%, 5.2% and 0.3% of 3076 genes altered unique to rWT, DEL, C57BL/6 and INS respectively following 1000 μg/kg TCDD). Hepatic Pxdc1 was significantly repressed in all three TCDD-sensitive animal models (C57BL/6 and rWT mice, and L-E rat) after TCDD exposure. Three genes, including Cxxc5, Sugp1 and Hgfac, demonstrated different AHRE-1 (full) motif occurrences within their promoter regions between rat strains, as well as different patterns of mRNA abundance. Several hepatic proteins showed parallel up- or downward alterations with their RNAs, with three genes (SNRK, IGTP and IMPA2) showing consistent, strain-dependent changes. These data show the value of integrating genomic, transcriptomic and proteomic evidence across multi-species models in toxicologic studies.

The International Society of Nephrology/Renal Pathology Society (ISN/RPS) lupus nephritis (LN) classification is under reconsideration, given challenges with inter-rater reliability and resultant inconsistent relationship with treatment response. Integration of molecular classifiers into histologic evaluation can improve diagnostic precision and identify therapeutic targets. This study described the relationship between histological and molecular phenotypes and clinical responses in LN. Renal compartmental mRNA abundance was measured in 54 biopsy specimens from LN patients and correlated to ISN/RPS classification and individual histologic lesions. A subset of transcripts was also evaluated in sequential biopsies of a separate longitudinal cohort of 36 patients with paired samples obtained at the time of flare and at follow up. Unsupervised clustering based on mRNA abundance did not demonstrate a relationship with the (ISN/RPS) classification, nor did univariate statistical analysis. Exploratory analyses suggested a correlation with individual histologic lesions. Glomerular FN1 (fibronectin), SPP1 (secreted phosphoprotein 1), and LGALS3 (galectin 3) abundance correlated with disease activity and changed following treatment. Exploratory analyses suggested relationships between specific transcripts and individual histologic lesions, with the important representation of interferon-regulated genes. Our findings suggested that the current LN classification could be refined by the inclusion of molecular descriptors. Combining molecular and pathologic kidney biopsy phenotypes may hold promise to better classify disease and identify actionable treatment targets and merits further exploration in larger cohorts.

Alternative splicing is a co-transcriptional mechanism that generates protein diversity by including or excluding exons in different combinations, thereby expanding the diversity of protein isoforms of a single gene. Abnormalities in this process can result in deleterious effects to human health, and several xenobiotics are known to interfere with splicing regulation through multiple mechanisms. These changes could lead to human diseases such as cancer, neurological disorders, autoimmune diseases, and developmental disorders. 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is an environmental contaminant generated as a byproduct of various industrial activities. Exposure to this dioxin has been linked to a wide range of pathologies through the alteration of multiple cellular processes. However, the effects of TCDD exposure on alternative splicing have not yet been studied. Here, we investigated whether a single po. dose of 5 μg/kg or 500 μg/kg TCDD influence hepatic alternative splicing in adult male C57BL/6Kou mouse. We identified several genes whose alternative splicing of precursor messenger RNAs was modified following TCDD exposure. In particular, we demonstrated that alternative splicing of Cyp1a1, Ahrr, and Actn1 was significantly altered after TCDD treatment. These findings show that the exposure to TCDD has an impact on alternative-splicing, and suggest a new avenue for understanding TCDD-mediated toxicity and pathogenesis., Competing Interests: The authors have declared that no competing interests exist.

Recent studies have revealed a possible link between the activities of polymorphic arylamine N-acetyltransferases (NATs) and energy metabolism. We used a Nat1/Nat2 double knockout (KO) mouse model to demonstrate that ablation of the two Nat genes is associated with modest, intermittent alterations in respiratory exchange rate. Pyruvate tolerance tests show that double KO mice have attenuated hepatic gluconeogenesis when maintained on a high-fat/high-sucrose diet. Absence of the two Nat genes also leads to an increase in the hepatic concentration of coenzyme A in mice fed a high-fat/high-sucrose diet. Our results suggest a modest involvement of NAT in energy metabolism in mice, which is consistent with the absence of major phenotypic deregulation of energy metabolism in slow human acetylators., (© 2019 Federation of European Biochemical Societies.)

IMA-08401 (C2) is a novel aryl hydrocarbon receptor (AHR) agonist and selective AHR modulator (SAHRM) that is structurally similar to laquinimod (LAQ). Both compounds are converted to the AHR-active metabolite DELAQ (IMA-06201) in vivo. SAHRMs have been proposed as therapeutic options for various autoimmune disorders. Clinical trials on LAQ have not reported any significant toxic outcomes and C2 has shown low toxicity in rats; however, their functional resemblance to the highly toxic AHR agonist 2,3,7,8-tetrachlorodibenzo- p -dioxin (TCDD) raises questions. Here, we characterize the hepatic transcriptomic changes induced by acute (single-dose) and subacute exposure (repeated dosing for 5 days followed by a 5-day recovery period) to C2 in Sprague-Dawley rats. Exposure to C2 leads to activation of the AHR, as shown by altered transcription of Cyp1a1 . We identify a heightened response early after exposure that drops off by day 10. Acute exposure to C2 leads to changes to transcription of genes involved in antiviral and antibacterial responses, which highlights the immunomodulator effects of this AHR agonist. Subacute exposure causes an oxidative stress response in the liver, the consequences of which require further study on target tissues such as the CNS and immune system, both of which may be compromised in this patient population.

Background: We introduce BPG, a framework for generating publication-quality, highly-customizable plots in the R statistical environment., Results: This open-source package includes multiple methods of displaying high-dimensional datasets and facilitates generation of complex multi-panel figures, making it suitable for complex datasets. A web-based interactive tool allows online figure customization, from which R code can be downloaded for integration with computational pipelines., Conclusion: BPG provides a new approach for linking interactive and scripted data visualization and is available at http://labs.oicr.on.ca/boutros-lab/software/bpg or via CRAN at https://cran.r-project.org/web/packages/BoutrosLab.plotting.general.

Smoking has historically been recognized as a negative prognostic factor in head and neck squamous cell carcinoma (HNSCC). This study aimed to assess the mutational differences between heavy smokers (>20 pack years) and never smokers among the HNSCC patients within The Cancer Genome Atlas (TCGA). Single nucleotide variation and copy number aberration differences between heavy smokers and never smokers were compared within human papillomavirus-positive (HPV-positive) (n = 67) and HPV-negative (n = 431) TCGA cohorts with HNSCC, and the impact of these mutations on survival were assessed. No genes were differentially mutated between smoking and never-smoking patients with HPV-positive tumors. By contrast, in HPV-negative tumors, NSD1 and COL1A11 were found to be more frequently mutated in heavy smokers, while CASP8 was more frequently altered in never smokers. HPV-negative patients with NSD1 mutations experienced significantly improved overall survival compared with NSD1 WT patients. This improved prognosis was validated in an independent cohort of 77 oral cavity cancer patients and a meta-analysis that included 2 additional data sets (688 total patients, hazard ratio for death 0.44, 95% CI, 0.30-0.65). NSD1 mutations are more common in HPV-negative heavy smokers, define a cohort with favorable prognosis, and may represent a clinically useful biomarker to guide treatment deintensification for HPV-negative patients.

Anaplastic thyroid cancer (ATC) is a rare and lethal human malignancy with no known effective therapies in the majority of cases. Despite the use of conventional treatments such as chemotherapy, radiation and surgical resection, this disease remains almost universally fatal. In the present study, we identified the JAK2 inhibitor Lestaurtinib as a potent compound when testing against 13 ATC cell lines. Lestaurtinib demonstrated a potent antiproliferative effect in vitro at nanomolar concentrations. Furthermore, Lestaurtinib impeded cell migration and the ability to form colonies from single cells using scratch-wound and colony formation assays, respectively. Flow cytometry was used for cell cycle analysis following drug treatment and demonstrated arrest at the G2/M phase of the cell cycle, indicative of a cytostatic effect. In vivo studies using the chick chorioallantoic membrane xenograft models demonstrated that treatment with Lestaurtinib resulted in a significant decrease in endpoint tumor volume and vascularity using power Doppler ultrasound imaging. Overall, this study provides evidence that Lestaurtinib is a potent antiproliferative agent with potential antiangiogenic activity that warrants further investigation as a targeted therapy for ATC., Competing Interests: The authors have declared that no competing interests exist.

Both a lack of biomarkers and relatively ineffective treatments constitute impediments to management of lupus nephritis (LN). Here we used gene expression microarrays to contrast the transcriptomic profiles of active SLE patients with and without LN to identify potential biomarkers for this condition. RNA isolated from whole peripheral blood of active SLE patients was used for transcriptomic profiling and the data analyzed by linear modeling, with corrections for multiple testing. Results were validated in a second cohort of SLE patients, using NanoString technology. The majority of genes demonstrating altered transcript abundance between patients with and without LN were neutrophil-related. Findings in the validation cohort confirmed this observation and showed that levels of RNA abundance in renal remission were similar to active patients without LN. In secondary analyses, RNA abundance correlated with disease activity, hematuria and proteinuria, but not renal biopsy changes. As abundance levels of the individual transcripts correlated strongly with each other, a composite neutrophil score was generated by summing all levels before examining additional correlations. There was a modest correlation between the neutrophil score and the blood neutrophil count, which was largely driven by the dose of glucocorticosteroids and not the proportion of low density and/or activated neutrophils. Analysis of longitudinal data revealed no correlation between baseline neutrophil score or changes over the first year of follow-up with subsequent renal flare or treatment outcomes, respectively. The findings argue that although the neutrophil score is associated with LN, its clinical utility as a biomarker may be limited.

Prostate tumours are highly variable in their response to therapies, but clinically available prognostic factors can explain only a fraction of this heterogeneity. Here we analysed 200 whole-genome sequences and 277 additional whole-exome sequences from localized, non-indolent prostate tumours with similar clinical risk profiles, and carried out RNA and methylation analyses in a subset. These tumours had a paucity of clinically actionable single nucleotide variants, unlike those seen in metastatic disease. Rather, a significant proportion of tumours harboured recurrent non-coding aberrations, large-scale genomic rearrangements, and alterations in which an inversion repressed transcription within its boundaries. Local hypermutation events were frequent, and correlated with specific genomic profiles. Numerous molecular aberrations were prognostic for disease recurrence, including several DNA methylation events, and a signature comprised of these aberrations outperformed well-described prognostic biomarkers. We suggest that intensified treatment of genomically aggressive localized prostate cancer may improve cure rates.

Background: 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is the most potent congener of the dioxin class of environmental contaminants. Exposure to TCDD causes a wide range of toxic outcomes, ranging from chloracne to acute lethality. The severity of toxicity is highly dependent on the aryl hydrocarbon receptor (AHR). Binding of TCDD to the AHR leads to changes in transcription of numerous genes. Studies evaluating the transcriptional changes brought on by TCDD may provide valuable insight into the role of the AHR in human health and disease. We therefore compiled a collection of transcriptomic datasets that can be used to aid the scientific community in better understanding the transcriptional effects of ligand-activated AHR., Results: Specifically, we have created a datasets package - TCDD.Transcriptomics - for the R statistical environment, consisting of 63 unique experiments comprising 377 samples, including various combinations of 3 species (human derived cell lines, mouse and rat), 4 tissue types (liver, kidney, white adipose tissue and hypothalamus) and a wide range of TCDD exposure times and doses. These datasets have been fully standardized using consistent preprocessing and annotation packages (available as of September 14, 2015). To demonstrate the utility of this R package, a subset of "AHR-core" genes were evaluated across the included datasets. Ahrr, Nqo1 and members of the Cyp family were significantly induced following exposure to TCDD across the studies as expected while Aldh3a1 was induced specifically in rat liver. Inmt was altered only in liver tissue and primarily by rat-AHR., Conclusions: Analysis of the "AHR-core" genes demonstrates a continued need for studies surrounding the impact of AHR-activity on the transcriptome; genes believed to be consistently regulated by ligand-activated AHR show surprisingly little overlap across species and tissues. Until now, a comprehensive assessment of the transcriptome across these studies was challenging due to differences in array platforms, processing methods and annotation versions. We believe that this package, which is freely available for download ( http://labs.oicr.on.ca/boutros-lab/tcdd-transcriptomics ) will prove to be a highly beneficial resource to the scientific community evaluating the effects of TCDD exposure as well as the variety of functions of the AHR.

2,3,7,8 Tetrachlorodibenzo-p-dioxin (TCDD) is an aromatic, long-lived environmental contaminant. While the pathogenesis of TCDD-induced toxicity is poorly understood, it has been shown that the aryl hydrocarbon receptor (AHR) is required. However, the specific transcriptomic changes that lead to toxic outcomes have not yet been identified. We previously identified a panel of 33 genes that respond to TCDD treatment in two TCDD-sensitive rodent species. To identify genes involved in the onset of hepatic toxicity, we explored 25 of these in-depth using liver from two rat strains: the TCDD-resistant Han/Wistar (H/W) and the TCDD-sensitive Long-Evans (L-E). Time course and dose-response analyses of mRNA abundance following TCDD insult indicate that eight genes are similarly regulated in livers of both strains of rat, suggesting that they are not central to the severe L-E-specific TCDD-induced toxicities. The remaining 17 genes exhibited various divergent mRNA abundances between L-E and H/W strains after TCDD treatment. Several genes displayed a biphasic response where the initial response to TCDD treatment was followed by a secondary response, usually of larger magnitude in L-E liver. This secondary response was most often an exaggeration of the original TCDD-induced response. Only cytochrome b5 type A (microsomal) (Cyb5a) had equivalent TCDD sensitivity to the prototypic AHR-responsive cytochrome P450, family 1, subfamily a, polypeptide 1 (Cyp1a1), while six genes were less sensitive. Four genes showed an early inter-strain difference that was sustained throughout most of the time course (atypical chemokine receptor 3 (Ackr3), collagen, type XVIII, alpha 1 (Col18a1), Cyb5a and glutamate dehydrogenase 1 (Glud1)), and of those genes examined in this study, are most likely to represent genes involved in the pathogenesis of TCDD-induced hepatotoxicity in L-E rats., Competing Interests: ABO has served as a paid consultant to The Dow Chemical Company as a member of their Dioxin Scientific Advisory Board. Other authors declare that they have no conflicts of interest.

Background: Management of lupus nephritis (LN) would be greatly aided by the discovery of biomarkers that accurately reflect changes in disease activity. Here, we used a proteomics approach to identify potential urinary biomarkers associated with LN., Methods: Urine was obtained from 60 LN patients with paired renal biopsies, 25 active non-LN SLE patients, and 24 healthy controls. Using Luminex, 128 analytes were quantified and normalized to urinary creatinine levels. Data were analyzed by linear modeling and non-parametric statistics, with corrections for multiple comparisons. A second cohort of 33 active LN, 16 active non-LN, and 30 remission LN SLE patients was used to validate the results., Results: Forty-four analytes were identified that were significantly increased in active LN as compared to active non-LN. This included a number of unique proteins (e.g., TIMP-1, PAI-1, PF4, vWF, and IL-15) as well as known candidate LN biomarkers (e.g., adiponectin, sVCAM-1, and IL-6), that differed markedly (>4-fold) between active LN and non-LN, all of which were confirmed in the validation cohort and normalized in remission LN patients. These proteins demonstrated an enhanced ability to discriminate between active LN and non-LN patients over several previously reported biomarkers. Ten proteins were found to significantly correlate with the activity score on renal biopsy, eight of which strongly discriminated between active proliferative and non-proliferative/chronic renal lesions., Conclusions: A number of promising urinary biomarkers that correlate with the presence of active renal disease and/or renal biopsy changes were identified and appear to outperform many of the existing proposed biomarkers.

Background: Visualization of data generated by high-throughput, high-dimensionality experiments is rapidly becoming a rate-limiting step in computational biology. There is an ongoing need to quickly develop high-quality visualizations that can be easily customized or incorporated into automated pipelines. This often requires an interface for manual plot modification, rapid cycles of tweaking visualization parameters, and the generation of graphics code. To facilitate this process for the generation of highly-customizable, high-resolution Venn and Euler diagrams, we introduce VennDiagramWeb: a web application for the widely used VennDiagram R package. VennDiagramWeb is hosted at http://venndiagram.res.oicr.on.ca/ ., Results: VennDiagramWeb allows real-time modification of Venn and Euler diagrams, with parameter setting through a web interface and immediate visualization of results. It allows customization of essentially all aspects of figures, but also supports integration into computational pipelines via download of R code. Users can upload data and download figures in a range of formats, and there is exhaustive support documentation., Conclusions: VennDiagramWeb allows the easy creation of Venn and Euler diagrams for computational biologists, and indeed many other fields. Its ability to support real-time graphics changes that are linked to downloadable code that can be integrated into automated pipelines will greatly facilitate the improved visualization of complex datasets. For application support please contact Paul.Boutros@oicr.on.ca.

Polychlorinated dibenzodioxins are environmental contaminants commonly produced as a by-product of industrial processes. The most potent of these, 2,3,7,8-tetrachlorodibenzo-ρ-dioxin (TCDD), is highly lipophilic, leading to bioaccumulation. White adipose tissue (WAT) is a major site for energy storage, and is one of the organs in which TCDD accumulates. In laboratory animals, exposure to TCDD causes numerous metabolic abnormalities, including a wasting syndrome. We therefore investigated the molecular effects of TCDD exposure on WAT by profiling the transcriptomic response of WAT to 100μg/kg of TCDD at 1 or 4days in TCDD-sensitive Long-Evans (Turku/AB; L-E) rats. A comparative analysis was conducted simultaneously in identically treated TCDD-resistant Han/Wistar (Kuopio; H/W) rats one day after exposure to the same dose. We sought to identify transcriptomic changes coinciding with the onset of toxicity, while gaining additional insight into later responses. More transcriptional responses to TCDD were observed at 4days than at 1day post-exposure, suggesting WAT shows mostly secondary responses. Two classic AHR-regulated genes, Cyp1a1 and Nqo1, were significantly induced by TCDD in both strains, while several genes involved in the immune response, including Ms4a7 and F13a1 were altered in L-E rats alone. We compared genes affected by TCDD in rat WAT and human adipose cells, and observed little overlap. Interestingly, very few genes involved in lipid metabolism exhibited altered expression levels despite the pronounced lipid mobilization from peripheral fat pads by TCDD in L-E rats. Of these genes, the lipolysis-associated Lpin1 was induced slightly over 2-fold in L-E rat WAT on day 4., (Copyright © 2015. Published by Elsevier Inc.)

Background: 2,3,7,8-tetrachlorodibenzo-p-dixion (TCDD) is the most potent of the dioxin congeners, capable of causing a wide range of toxic effects across numerous animal models. Previous studies have demonstrated that males and females of the same species can display divergent sensitivity phenotypes to TCDD toxicities. Although it is now clear that most TCDD-induced toxic outcomes are mediated by the aryl hydrocarbon receptor (AHR), the mechanism of differential responses to TCDD exposure between sexes remains largely unknown. To investigate the differential sensitivities in male and female mice, we profiled the hepatic transcriptomic responses 4 days following exposure to various amounts of TCDD (125, 250, 500 or 1000 μg/kg) in adult male and female C57BL/6Kuo mice., Results: Several key findings were revealed by our study. 1) Hepatic transcriptomes varied significantly between the sexes at all doses examined. 2) The liver transcriptome of males was more dysregulated by TCDD than that of females. 3) The alteration of "AHR-core" genes was consistent in magnitude, regardless of sex. 4) A subset of genes demonstrated sex-dependent TCDD-induced transcriptional changes, including Fmo3 and Nr1i3, which were significantly induced in livers of male mice only. In addition, a meta-analysis was performed to contrast transcriptomic profiles of various organisms and tissues following exposure to equitoxic doses of TCDD. Minimal overlap was observed in the differences between TCDD-sensitive or TCDD-resistant models., Conclusions: Sex-dependent sensitivities to TCDD exposure are associated with a set of sex-specific TCDD-responsive genes. In addition, complex interactions between the aryl hydrocarbon and sex hormone receptors may affect the observable differences in sensitivity phenotypes between the sexes. Further work is necessary to better understand the roles of those genes altered by TCDD in a sex-dependent manner, and their association with changes to sex hormones and receptors.

Herein we provide a detailed molecular analysis of the spatial heterogeneity of clinically localized, multifocal prostate cancer to delineate new oncogenes or tumor suppressors. We initially determined the copy number aberration (CNA) profiles of 74 patients with index tumors of Gleason score 7. Of these, 5 patients were subjected to whole-genome sequencing using DNA quantities achievable in diagnostic biopsies, with detailed spatial sampling of 23 distinct tumor regions to assess intraprostatic heterogeneity in focal genomics. Multifocal tumors are highly heterogeneous for single-nucleotide variants (SNVs), CNAs and genomic rearrangements. We identified and validated a new recurrent amplification of MYCL, which is associated with TP53 deletion and unique profiles of DNA damage and transcriptional dysregulation. Moreover, we demonstrate divergent tumor evolution in multifocal cancer and, in some cases, tumors of independent clonal origin. These data represent the first systematic relation of intraprostatic genomic heterogeneity to predicted clinical outcome and inform the development of novel biomarkers that reflect individual prognosis.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is an environmental contaminant that produces myriad toxicities in most mammals. In rodents alone, there is a huge divergence in the toxicological response across species, as well as among different strains within a species. But there are also significant differences between males and females animals of a single strain. These differences are inconsistent across model systems: the severity of toxicity is greater in female rats than males, while male mice and guinea pigs are more sensitive than females. Because the specific events that underlie this difference remain unclear, we characterized the hepatic transcriptional response of adult male and female C57BL/6 mice to 500μg/kg TCDD at multiple time-points. The transcriptional profile diverged significantly between the sexes. Female mice demonstrated a large number of altered transcripts as early as 6h following treatment, suggesting a large primary response. Conversely, male animals showed the greatest TCDD-mediated response 144h following exposure, potentially implicating significant secondary responses. Nr1i3 was statistically significantly induced at all time-points in the sensitive male animals. This mRNA encodes the constitutive androstane receptor (CAR), a transcription factor involved in the regulation of xenobiotic metabolism, lipid metabolism, cell cycle and apoptosis. Surprisingly though, changes at the protein level (aside from the positive control, CYP1A1) were modest, with only FMO3 showing clear induction, and no genes with sex-differences. Thus, while male and female mice show transcriptional differences in their response to TCDD, their association with TCDD-induced toxicities remains unclear., (Copyright © 2015. Published by Elsevier Inc.)

Objective: The aim of this study was to determine whether anti-nucleosome antibodies function as activity-specific biomarkers in SLE., Methods: Fifty-one patients were recruited and followed prospectively with periodic clinical and biochemical assessments over a 14-month period. Disease activity was determined by the SLEDAI-2K. Anti-nucleosome antibody levels were measured by an ELISA and its utility as an activity-specific biomarker as compared with that of anti-dsDNA antibodies and C3 was assessed both at baseline and in longitudinal analysis., Results: Anti-nucleosome antibodies were significantly elevated in SLE patients vs controls and showed a moderate positive correlation with disease activity. The utility of anti-nucleosome antibodies in identifying patients with active disease in a cross-sectional analysis was comparable to that of anti-dsDNA antibodies and C3. Analysis of variance demonstrated that the level of anti-nucleosome antibodies and C3 varied significantly with changes in disease activity over time. Changes in clinical state were not mirrored by changes in anti-dsDNA antibodies. In time-dependent analysis, anti-nucleosome antibodies showed a better fit over time than anti-dsDNA antibodies and C3. In pairwise comparisons, C3 and anti-nucleosome antibodies outperformed other models, including the conventional pairing of C3 and anti-dsDNA antibodies, however, no biomarker alone or as a group accurately predicted impending remissions or exacerbations., Conclusion: Anti-nucleosome antibodies demonstrate greater fidelity as a biomarker for changes in SLE disease activity than traditional biomarkers, supporting the routine monitoring of this antibody in clinical practice., (© The Author 2014. Published by Oxford University Press on behalf of the British Society for Rheumatology. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

In some mammals, halogenated aromatic hydrocarbon (HAH) exposure causes wasting syndrome, defined as significant weight loss associated with lethal outcomes. The most potent HAH in causing wasting is 2,3,7,8-tetrachlorodibenzo-ρ-dioxin (TCDD), which exerts its toxic effects through the aryl hydrocarbon receptor (AHR). Since TCDD toxicity is thought to predominantly arise from dysregulation of AHR-transcribed genes, it was hypothesized that wasting syndrome is a result of to TCDD-induced dysregulation of genes involved in regulation of food-intake. As the hypothalamus is the central nervous systems' regulatory center for food-intake and energy balance. Therefore, mRNA abundances in hypothalamic tissue from two rat strains with widely differing sensitivities to TCDD-induced wasting syndrome: TCDD-sensitive Long-Evans rats and TCDD-resistant Han/Wistar rats, 23h after exposure to TCDD (100μg/kg) or corn oil vehicle. TCDD exposure caused minimal transcriptional dysregulation in the hypothalamus, with only 6 genes significantly altered in Long-Evans rats and 15 genes in Han/Wistar rats. Two of the most dysregulated genes were Cyp1a1 and Nqo1, which are induced by TCDD across a wide range of tissues and are considered sensitive markers of TCDD exposure. The minimal response of the hypothalamic transcriptome to a lethal dose of TCDD at an early time-point suggests that the hypothalamus is not the predominant site of initial events leading to hypophagia and associated wasting. TCDD may affect feeding behaviour via events upstream or downstream of the hypothalamus, and further work is required to evaluate this at the level of individual hypothalamic nuclei and subregions., (Copyright © 2014 The Authors. Published by Elsevier Ireland Ltd.. All rights reserved.)

Background: Research on the aryl hydrocarbon receptor (AHR) has largely focused on variations in toxic outcomes resulting from its activation by halogenated aromatic hydrocarbons. But the AHR also plays key roles in regulating pathways critical for development, and after decades of research the mechanisms underlying physiological regulation by the AHR remain poorly characterized. Previous studies identified several core genes that respond to xenobiotic AHR ligands across a broad range of species and tissues. However, only limited inferences have been made regarding its role in regulating constitutive gene activity, i.e. in the absence of exogenous ligands. To address this, we profiled transcriptomic variations between AHR-active and AHR-less-active animals in the absence of an exogenous agonist across five tissues, three of which came from rats (hypothalamus, white adipose and liver) and two of which came from mice (kidney and liver). Because AHR status alone has been shown sufficient to alter transcriptomic responses, we reason that by contrasting profiles amongst AHR-variant animals, we may elucidate effects of the AHR on constitutive mRNA abundances., Results: We found significantly more overlap in constitutive mRNA abundances amongst tissues within the same species than from tissues between species and identified 13 genes (Agt, Car3, Creg1, Ctsc, E2f6, Enpp1, Gatm, Gstm4, Kcnj8, Me1, Pdk1, Slc35a3, and Sqrdl) that are affected by AHR-status in four of five tissues. One gene, Creg1, was significantly up-regulated in all AHR-less-active animals. We also find greater overlap between tissues at the pathway level than at the gene level, suggesting coherency to the AHR signalling response within these processes. Analysis of regulatory motifs suggests that the AHR mostly mediates transcriptional regulation via direct binding to response elements., Conclusions: These findings, though preliminary, present a platform for further evaluating the role of the AHR in regulation of constitutive mRNA levels and physiologic function.

Western blotting is a well-established, inexpensive and accurate way of measuring protein content. Because of technical variation between wells, normalization is required for valid interpretation of results across multiple samples. Typically this involves the use of one or more endogenous controls to adjust the measured levels of experimental molecules. Although some endogenous controls are widely used, validation is required for each experimental system. This is critical when studying transcriptional-modulators, such as toxicants like 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD).To address this issue, we examined hepatic tissue from 192 mice representing 47 unique combinations of strain, sex, Ahr-genotype, TCDD dose and treatment time. We examined 7 candidate reference proteins in each animal and assessed consistency of protein abundance through: 1) TCDD-induced fold-difference in protein content from basal levels, 2) inter- and intra- animal stability, and 3) the ability of each candidate to reduce instability of the other candidates. Univariate analyses identified HPRT as the most stable protein. Multivariate analysis indicated that stability generally increased with the number of proteins used, but gains from using >3 proteins were small. Lastly, by comparing these new data to our previous studies of mRNA controls on the same animals, we were able to show that the ideal mRNA and protein control-genes are distinct, and use of only 2-3 proteins provides strong stability, unlike in mRNA studies in the same cohort, where larger control-gene batteries were needed.

Despite several decades of research, the complete mechanism by which 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and other xenobiotic agonists of the aryl hydrocarbon receptor (AHR) cause toxicity remains unclear. While it has been shown that the AHR is required for all major manifestations of toxicity, the specific downstream changes involved in the development of toxic phenotypes remain unknown. Here we examine a panel of 13 genes that are AHR-regulated in many species and tissues. We profiled their hepatic mRNA abundances in two rat strains with very different sensitivities to TCDD: the TCDD-sensitive Long-Evans (Turku/AB; L-E) and the TCDD-resistant Han/Wistar (Kuopio; H/W). We evaluated doses ranging from 0 to 3000μg/kg at 19h after TCDD exposure and time points ranging from 1.5 to 384h after exposure to 100μg/kg TCDD. Twelve of 13 genes responded to TCDD in at least one strain, and seven of these showed statistically significant inter-strain differences in the time course analysis (Aldh3a1, Cyp1a2, Cyp1b1, Cyp2a1, Fmo1, Nfe2l2 and Nqo1). Cyp2s1 did not respond to TCDD in either rat strain. Five genes exhibited biphasic responses to TCDD insult (Ahrr, Aldh3a1, Cyp1b1, Nfe2l2 and Nqo1), suggesting a secondary event, such as association with additional transcriptional modulators. Of the 12 genes that responded to TCDD during the dose-response analysis, none had an ED50 equivalent to that of Cyp1a1, the most sensitive gene in this study, while nine genes responded to doses at least 10-100 fold higher, in at least one strain (Ahrr (L-E), Aldh3a1 (both), Cyp1a2 (both), Cyp1b1 (both), Cyp2a1 (L-E), Inmt (both), Nfe2l2 (L-E), Nqo1 (L-E) and Tiparp (both)). These data shed new light on the association of the AHR target genes with TCDD toxicity, and in particular the seven genes exhibiting strain-specific differences represent strong candidate mediators of Type-II toxicities., (Copyright © 2013. Published by Elsevier Inc.)

Animal studies suggest temporary renin-angiotensin system (RAS) blockade enhances long-term vascular protective effects; however, this is not established in humans. Here we evaluated the long-term effects of prior RAS blockade on hemodynamic function, urinary measures of inflammation, and tissue antioxidant mRNA expression in patients with type 1 diabetes mellitus (T1DM) who participated in the 5-year Renin Angiotensin System Study (RASS). At 4 years after completing the RASS and discontinuing study medication, renal hemodynamic responses to clamped hyperglycemia were significantly greater in 18 patients in the RAS blockade group compared to 9 patients of the placebo-treated group. Individuals who had received RAS blockade also exhibited higher flow-mediated vasodilatation, reduced urinary cytokine excretion in response to hyperglycemia, and increased skin mRNA expression of superoxide dismutase-1 and catalase. Thus, patients with uncomplicated T1DM who received prior RAS blockade for 5 years maintain long-term effects on renal hemodynamic and systemic vascular function, inflammatory pathways in the kidney, and antioxidant enzyme expression in skin 4 years after discontinuation of therapy. Our findings suggest that sustained long-term protective effects of finite RAS inhibition requires further study.

Background: Quantitative real-time PCR (qPCR) is the "gold-standard" technique for measuring mRNA abundances. To correctly compare samples and generate biologically valid results, qPCR data usually require comprehensive normalization to account for sample content variation between reactions. The most common normalization approaches use one or more endogenous controls (reference or house-keeping genes) to adjust the measured levels of experimental genes appropriately. Ideal reference genes are those that display minimal variation across experimental conditions, and thus can vary widely across different biological systems. In particular, toxicogenomic studies of transcriptionally-disruptive toxins, like 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), require careful consideration of reference genes., Results: We examined seven candidate reference genes in 199 mice varying in genotype and time/dose of TCDD exposure. We assessed gene-stability in four ways: (1) the variance of the raw Cq values across biological replicates, (2) the fold-change from basal mRNA levels following treatment, (3) the inter- and intra-group stability evaluated using the NormFinder algorithm, (4) the comparative ΔCq method for each candidate gene. Univariate analyses showed Hprt and Eef1a1 are the two most stable individual reference genes. It has been suggested that using multiple genes would produce a more consistent normalization factor; multivariate analysis was performed using NormFinder. In general, stability increased with the number of genes used, but specific gene-combinations synergized., Conclusions: We have validated seven reference genes for use in analyzing mRNA abundances in mouse models of TCDD toxicity. The use of multiple reference genes increases stability, providing more consistent normalization and more reliable results. The number of reference genes used should be maximized, based on experimental capabilities (platform, sample availability, etc.). Our results show the benefit of validating reference genes using multiple methods prior to generating large biological datasets., (Copyright © 2013 The Authors. Published by Elsevier Ireland Ltd.. All rights reserved.)

Profiling of mRNA abundances with high-throughput platforms such as microarrays and RNA-seq has become an important tool in both basic and biomedical research. However, these platforms remain prone to systematic errors and have challenges in clinical and industrial applications. As a result, it is standard practice to validate a subset of key results using alternate technologies. Similarly, clinical and industrial applications typically involve transitions from a high-throughput discovery platform to medium-throughput validation ones. These medium-throughput validation platforms have high technical reproducibility and reduced sample input needs, and low sensitivity to sample quality (e.g., for processing FFPE specimens). Unfortunately, while medium-throughput platforms have proliferated, there are no comprehensive comparisons of them. Here we fill that gap by comparing two key medium-throughput platforms--NanoString's nCounter Analysis System and ABI's OpenArray System--to gold-standard quantitative real-time RT-PCR. We quantified 38 genes and positive and negative controls in 165 samples. Signal:noise ratios, correlations, dynamic range, and detection accuracy were compared across platforms. All three measurement technologies showed good concordance, but with divergent price/time/sensitivity trade-offs. This study provides the first detailed comparison of medium-throughput RNA quantification platforms and provides a template and a standard data set for the evaluation of additional technologies.

The biochemical and toxic effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) have been the subject of intense study for decades. It is now clear that essentially all TCDD-induced toxicities are mediated by DNA-protein interactions involving the Aryl Hydrocarbon Receptor (AHR). Nevertheless, it remains unknown which AHR target genes cause TCDD toxicities. Several groups, including our own, have developed rodent model systems to probe these questions. mRNA expression profiling of these model systems has revealed significant inter-species heterogeneity in rodent hepatic responses to TCDD. It has remained unclear if this variability also exists within a species, amongst rodent strains. To resolve this question, we profiled the hepatic transcriptomic response to TCDD of diverse rat strains (L-E, H/W, F344 and Wistar rats) and two lines derived from L-E×H/W crosses, at consistent age, sex, and dosing (100 μg/kg TCDD for 19 h). Using this uniquely consistent dataset, we show that the majority of TCDD-induced alterations in mRNA abundance are strain/line-specific: only 11 genes were affected by TCDD across all strains, including well-known dioxin-responsive genes such as Cyp1a1 and Nqo1. Our analysis identified two novel universally dioxin-responsive genes as well as 4 genes induced by TCDD in dioxin-sensitive rats only. These 6 genes are strong candidates to explain TCDD-related toxicities, so we validated them using 152 animals in time-course (0 to 384 h) and dose-response (0 to 3000 μg/kg) experiments. This study reveals that different rat strains exhibit dramatic transcriptional heterogeneity in their hepatic responses to TCDD and that inter-strain comparisons can help identify candidate toxicity-related genes., (Copyright © 2012 Elsevier Inc. All rights reserved.)

The dioxin congener 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) causes a wide range of toxic effects in rodent species, all of which are mediated by a ligand-dependent transcription-factor, the aryl hydrocarbon receptor (AHR). The Han/Wistar (Kuopio) (H/W) strain shows exceptional resistance to many TCDD-induced toxicities; the LD₅₀ of > 9600 μg/kg for H/W rats is higher than for any other wild-type mammal known. We previously showed that this resistance primarily results from H/W rats expressing a variant AHR isoform that has a substantial portion of the AHR transactivation domain deleted. Despite this large deletion, H/W rats are not entirely refractory to the effects of TCDD; the variant AHR in these animals remains fully competent to up-regulate well-known dioxin-inducible genes. TCDD-sensitive (Long-Evans, L-E) and resistant (H/W) rats were treated with either corn-oil (with or without feed-restriction) or 100 μg/kg TCDD for either four or ten days. Hepatic transcriptional profiling was done using microarrays, and was validated by RT-PCR analysis of 41 genes. A core set of genes was altered in both strains at all time points tested, including CYP1A1, CYP1A2, CYP1B1, Nqo1, Aldh3a1, Tiparp, Exoc3, and Inmt. Outside this core, the strains differed significantly in the breadth of response: three-fold more genes were altered in L-E than H/W rats. At ten days almost all expressed genes were dysregulated in L-E rats, likely reflecting emerging toxic responses. Far fewer genes were affected by feed-restriction, suggesting that only a minority of the TCDD-induced changes are secondary to the wasting syndrome., (Copyright © 2010 Elsevier Inc. All rights reserved.)