Development and validation of the TGx-HDACi transcriptomic biomarker to detect histone deacetylase inhibitors in human TK6 cells.

Transcriptomic biomarkers can be used to inform molecular initiating and key events involved in a toxicant's mode of action. To address the limited approaches available for identifying epigenotoxicants, we developed and assessed a transcriptomic biomarker of histone deacetylase inhibition (HDACi). First, we assembled a set of ten prototypical HDACi and ten non-HDACi reference compounds. Concentration-response experiments were performed for each chemical to collect TK6 human lymphoblastoid cell samples after 4 h of exposure and to assess cell viability following a 20-h recovery period in fresh media. One concentration was selected for each chemical for whole transcriptome profiling and transcriptomic signature derivation, based on cell viability at the 24-h time point and on maximal induction of HDACi-response genes (RGL1, NEU1, GPR183) or cellular stress-response genes (ATF3, CDKN1A, GADD45A) analyzed by TaqMan qPCR assays after 4 h of exposure. Whole transcriptomes were profiled after 4 h exposures by Templated Oligo-Sequencing (TempO-Seq). By applying the nearest shrunken centroid (NSC) method to the whole transcriptome profiles of the reference compounds, we derived an 81-gene toxicogenomic (TGx) signature, referred to as TGx-HDACi, that classified all 20 reference compounds correctly using NSC classification and the Running Fisher test. An additional 4 HDACi and 7 non-HDACi were profiled and analyzed using TGx-HDACi to further assess classification performance; the biomarker accurately classified all 11 compounds, including 3 non-HDACi epigenotoxicants, suggesting a promising specificity toward HDACi. The availability of TGx-HDACi increases the diversity of tools that can facilitate mode of action analysis of toxicants using gene expression profiling.

Arsenite and cadmium promote the development of mammary tumors.

Previous studies demonstrate that the heavy metal cadmium and the metalloid arsenite activate estrogen receptor-alpha in breast cancer cells by forming a high-affinity complex with the ligand-binding domain of the receptor and that environmentally relevant doses of cadmium have estrogen-like activity in vivo. The present study showed that in estrogen-receptor positive cells, arsenite and cadmium increased the global expression of estrogen-responsive genes and that an environmentally relevant dose of arsenite also had estrogen-like activity in vivo. Similar to estrogens, exposure of ovariectomized animals to arsenite induced the expression of the progesterone receptor, GREB1, and c-fos in the mammary gland and the expression of complement C3, c-fos, and cyclin D1 in the uterus and the increase was blocked by the antiestrogen ICI-182,780. When virgin female animals were fed a diet, that mimics exposure to either arsenite or cadmium, and challenged with the chemical carcinogen dimethylbenzanthracene, there was an increase in the incidence of mammary tumors and a decrease in the time to tumor onset, but no difference in the total number of tumors, tumor multiplicity, or total tumor volume. Together with published results, these data showed that environmentally relevant amounts of arsenite and cadmium had estrogen-like activity in vivo and promoted mammary tumorigenesis.

Development and validation of a high-throughput transcriptomic biomarker to address 21st century genetic toxicology needs.

Interpretation of positive genotoxicity findings using the current in vitro testing battery is a major challenge to industry and regulatory agencies. These tests, especially mammalian cell assays, have high sensitivity but suffer from low specificity, leading to high rates of irrelevant positive findings (i.e., positive results in vitro that are not relevant to human cancer hazard). We developed an in vitro transcriptomic biomarker-based approach that provides biological relevance to positive genotoxicity assay data, particularly for in vitro chromosome damage assays, and propose its application for assessing the relevance of the in vitro positive results to carcinogenic hazard. The transcriptomic biomarker TGx-DDI (previously known as TGx-28.65) readily distinguishes DNA damage-inducing (DDI) agents from non-DDI agents. In this study, we demonstrated the ability of the biomarker to classify 45 test agents across a broad set of chemical classes as DDI or non-DDI. Furthermore, we assessed the biomarker's utility in derisking known irrelevant positive agents and evaluated its performance across analytical platforms. We correctly classified 90% (9 of 10) of chemicals with irrelevant positive findings in in vitro chromosome damage assays as negative. We developed a standardized experimental and analytical protocol for our transcriptomics biomarker, as well as an enhanced application of TGx-DDI for high-throughput cell-based genotoxicity testing using nCounter technology. This biomarker can be integrated in genetic hazard assessment as a follow-up to positive chromosome damage findings. In addition, we propose how it might be used in chemical screening and assessment. This approach offers an opportunity to significantly improve risk assessment and reduce cost.

Modulation of fatty acid and bile acid metabolism by peroxisome proliferator-activated receptor α protects against alcoholic liver disease.

BACKGROUND: Chronic alcohol intake affects liver function and causes hepatic pathological changes. It has been shown that peroxisome proliferator-activated receptor α (PPARα)-null mice developed more pronounced hepatic changes than wild-type (WT) mice after chronic exposure to a diet containing 4% alcohol. The remarkable similarity between the histopathology of alcoholic liver disease (ALD) in Ppara-null model and in humans, and the fact that PPARα expression and activity in human liver are less than one-tenth of those in WT mouse liver make Ppara-null a good system to investigate ALD. METHODS: In this study, the Ppara-null model was used to elucidate the dynamic regulation of PPARα activity during chronic alcohol intake. Hepatic transcriptomic and metabolomic analyses were used to examine alterations of gene expression and metabolites associated with pathological changes. The changes triggered by alcohol consumption on gene expression and metabolites in Ppara-null mice were compared with those in WT mice. RESULTS: The results showed that in the presence of PPARα, 3 major metabolic pathways in mitochondria, namely the fatty acid ß-oxidation, the tricarboxylic acid cycle, and the electron transfer chain, were induced in response to a 2-month alcohol feeding, while these responses were greatly reduced in the absence of PPARα. In line with the transcriptional modulations of these metabolic pathways, a progressive accumulation of triglycerides, a robust increase in hepatic cholic acid and its derivatives, and a strong induction of fibrogenesis genes were observed exclusively in alcohol-fed Ppara-null mice. CONCLUSIONS: These observations indicate that PPARα plays a protective role to enhance mitochondrial function in response to chronic alcohol consumption by adaptive transcriptional activation and suggest that activation of this nuclear receptor may be of therapeutic value in the treatment for ALD.

FAILLA MEMORIAL LECTURE How We Got Here: One Laboratory's Odyssey in the Field of Radiation-Inducible Genes.

This review focuses on early discoveries that contributed to our understanding and the scope of transcriptional responses after radiation damage. Before the development of modern approaches to assess overall global transcriptomic responses, the idea that mammalian cells could respond to DNA-damaging agents in a manner analogous to bacteria was not generally accepted. To investigate this possibility, the development of technology to identify differentially expressed low-abundance transcripts substantially facilitated our appreciation that DNA damaging agents like UV radiation and subsequently ionizing radiation did in fact produce robust transcriptional responses. Here we focus on our identification and characterization of radiation-inducible genes, and how even early studies on stress gene signaling highlighted the broad scope of transcriptional responses to radiation damage. Since then, the central role of transcriptional responses to radiation injury in maintaining genome integrity has been highlighted in many processes, including cell cycle checkpoint control, resistance to cancer by p53 and other key factors, cell senescence, and metabolism.

Host microbiome depletion attenuates biofluid metabolite responses following radiation exposure.

Development of novel biodosimetry assays and medical countermeasures is needed to obtain a level of radiation preparedness in the event of malicious or accidental mass exposures to ionizing radiation (IR). For biodosimetry, metabolic profiling with mass spectrometry (MS) platforms has identified several small molecules in easily accessible biofluids that are promising for dose reconstruction. As our microbiome has profound effects on biofluid metabolite composition, it is of interest how variation in the host microbiome may affect metabolomics based biodosimetry. Here, we 'knocked out' the microbiome of male and female C57BL/6 mice (Abx mice) using antibiotics and then irradiated (0, 3, or 8 Gy) them to determine the role of the host microbiome on biofluid radiation signatures (1 and 3 d urine, 3 d serum). Biofluid metabolite levels were compared to a sham and irradiated group of mice with a normal microbiome (Abx-con mice). To compare post-irradiation effects in urine, we calculated the Spearman's correlation coefficients of metabolite levels with radiation dose. For selected metabolites of interest, we performed more detailed analyses using linear mixed effect models to determine the effects of radiation dose, time, and microbiome depletion. Serum metabolite levels were compared using an ANOVA. Several metabolites were affected after antibiotic administration in the tryptophan and amino acid pathways, sterol hormone, xenobiotic and bile acid pathways (urine) and lipid metabolism (serum), with a post-irradiation attenuative effect observed for Abx mice. In urine, dose×time interactions were supported for a defined radiation metabolite panel (carnitine, hexosamine-valine-isoleucine [Hex-V-I], creatine, citric acid, and Nε,Nε,Nε-trimethyllysine [TML]) and dose for N1-acetylspermidine, which also provided excellent (AUROC ≥ 0.90) to good (AUROC ≥ 0.80) sensitivity and specificity according to the area under the receiver operator characteristic curve (AUROC) analysis. In serum, a panel consisting of carnitine, citric acid, lysophosphatidylcholine (LysoPC) (14:0), LysoPC (20:3), and LysoPC (22:5) also gave excellent to good sensitivity and specificity for identifying post-irradiated individuals at 3 d. Although the microbiome affected the basal levels and/or post-irradiation levels of these metabolites, their utility in dose reconstruction irrespective of microbiome status is encouraging for the use of metabolomics as a novel biodosimetry assay.

Identification of serum insulin-like growth factor binding protein 1 as diagnostic biomarker for early-stage alcohol-induced liver disease.

BACKGROUND: Alcohol consumption is a major cause of liver disease in humans. The use and monitoring of biomarkers associated with early, pre-clinical stages of alcohol-induced liver disease (pre-ALD) could facilitate diagnosis and treatment, leading to improved outcomes. METHODS: We investigated the pathological, transcriptomic and protein changes in early stages of pre-ALD in mice fed the Lieber-Decarli liquid diet with or without alcohol for four months to identify biomarkers for the early stage of alcohol induced liver injury. Mice were sampled after 1, 2 and 4 months treatment. RESULTS: Pathological examination revealed a modest increase in fatty liver changes in alcohol-treated mice. Transcriptomics revealed gene alterations at all time points. Most notably, the Igfbp1 (Insulin-Like Growth Factor Binding Protein 1) was selected as the best candidate gene for early detection of liver damage since it showed early and continuously enhanced induction during the treatment course. Consistent with the microarray data, both Igfbp1mRNA expression in the liver tissue and the IGFBP1 serum protein levels showed progressive and significant increases over the course of pre-ALD development. CONCLUSIONS: The results suggest that in conjunction with other tests, serum IGFBPI protein could provide an easily measured biomarker for early detection of alcohol-induced liver injury in humans.

Circulating cell-free methylated DNA reveals tissue-specific, cellular damage from radiation treatment.

Radiation therapy is an effective cancer treatment, although damage to healthy tissues is common. Here we analyzed cell-free, methylated DNA released from dying cells into the circulation to evaluate radiation-induced cellular damage in different tissues. To map the circulating DNA fragments to human and mouse tissues, we established sequencing-based, cell-type-specific reference DNA methylation atlases. We found that cell-type-specific DNA blocks were mostly hypomethylated and located within signature genes of cellular identity. Cell-free DNA fragments were captured from serum samples by hybridization to CpG-rich DNA panels and mapped to the DNA methylation atlases. In a mouse model, thoracic radiation-induced tissue damage was reflected by dose-dependent increases in lung endothelial and cardiomyocyte methylated DNA in serum. The analysis of serum samples from patients with breast cancer undergoing radiation treatment revealed distinct dose-dependent and tissue-specific epithelial and endothelial responses to radiation across multiple organs. Strikingly, patients treated for right-sided breast cancers also showed increased hepatocyte and liver endothelial DNA in the circulation, indicating the impact on liver tissues. Thus, changes in cell-free methylated DNA can uncover cell-type-specific effects of radiation and provide a readout of the biologically effective radiation dose received by healthy tissues.

PTEN tumor suppressor regulates p53 protein levels and activity through phosphatase-dependent and -independent mechanisms.

We show in this study that PTEN regulates p53 protein levels and transcriptional activity through both phosphatase-dependent and -independent mechanisms. The onset of tumor development in p53(+/-);Pten(+/-) mice is similar to p53(-/-) animals, and p53 protein levels are dramatically reduced in Pten(-/-) cells and tissues. Reintroducing wild-type or phosphatase-dead PTEN mutants leads to a significant increase in p53 stability. PTEN also physically associates with endogenous p53. Finally, PTEN regulates the transcriptional activity of p53 by modulating its DNA binding activity. This study provides a novel mechanism by which the loss of PTEN can functionally control "two" hits in the course of tumor development by concurrently modulating p53 activity.

A high-throughput and highly automated genotoxicity screening assay.

The increasing number of compounds under development and chemicals in commerce that require safety assessments pose a serious challenge for regulatory agencies worldwide. In vitro screening using toxicogenomic biomarkers has been proposed as a first-tier screen in chemical assessment and has been endorsed internationally. We previously developed, evaluated, and validated an in vitro transcriptomic biomarker responsive to DNA damage-inducing (DDI) agents, namely TGx-DDI, for genotoxicity testing in human cells and demonstrated the feasibility of using TGx-DDI in a medium-throughput, cell-based genotoxicity testing system by implementing this biomarker with the Nanostring nCounter system. In this current study, we took advantage of Nanostring nCounter Plexset technology to develop a highly auto­mated, multiplexed, and high-throughput genotoxicity testing assay, designated the TGx-DDI Plexset assay, which can increase the screening efficiency eight-fold compared to standard nCounter technology while decreasing the hands-on time. We demonstrate the high-throughput capability of this assay by eliminating concentration determination and RNA extraction steps without compromising the specificity and sensitivity of TGx-DDI. Thus, we propose that this simple, highly automated, multiplexed high-throughput pipeline can be widely used in chemical screening and assessment.

Small Molecule Signatures of Mice Lacking T-cell p38 Alternate Activation, a Model for Immunosuppression Conditions, after Total-Body Irradiation.

Several diagnostic biodosimetry tools have been in development that may aid in radiological/nuclear emergency responses. Of these, correlating changes in non-invasive biofluid small-molecule signatures to tissue damage from ionizing radiation exposure show promise for inclusion in predictive biodosimetry models. Integral to dose reconstruction has been determining how genotypic variation in the general population will affect model performance. Here, we used a mouse model that lacks the T-cell receptor specific alternative p38 pathway [p38αßY323F, double knock-in (DKI) mice] to determine how attenuated autoimmune and inflammatory responses may affect dose reconstruction. We exposed adult male DKI mice (8-10 weeks old) to 2 and 7 Gy in parallel with wild-type mice and assessed perturbations in urine (days 1, 3, 7) and serum (day 1) using a global metabolomics approach. A multidimensional scaling plot showed excellent separation of radiation-exposed groups in wild-type mice with slightly dampened responses in DKI mice. Validated metabolite panels were developed for urine [N6,N6,N6-trimethyllysine (TML), N1-acetylspermidine, spermidine, carnitine, acylcarnitine C21H35NO5, aminohippuric acid] and serum [phenylalanine, glutamine, propionylcarnitine, lysophosphatidylcholine (LysoPC 14:0), LysoPC (22:5)] to determine the area under the receiver operating characteristic curve (AUROC). For both urine and serum, excellent sensitivity and specificity (AUROC > 0.90) was observed for 0 Gy vs. 7 Gy groups irrespective of genotype using identical metabolite panels. Similarly, excellent to fair classification (AUROC > 0.75) was observed for ≤2 Gy vs. 7 Gy mice for both genotypes, however, model performance declined (AUROC < 0.75) between genotypes after irradiation. Overall, these results suggest immunosuppression should not compromise small molecule multiplex panels used in dose reconstruction for biodosimetry.

Integrated Genotoxicity Testing of three anti-infective drugs using the TGx-DDI transcriptomic biomarker and high-throughput CometChip® assay in TK6 cells.

Genotoxicity testing relies on the detection of gene mutations and chromosome damage and has been used in the genetic safety assessment of drugs and chemicals for decades. However, the results of standard genotoxicity tests are often difficult to interpret due to lack of mode of action information. The TGx-DDI transcriptomic biomarker provides mechanistic information on the DNA damage-inducing (DDI) capability of chemicals to aid in the interpretation of positive in vitro genotoxicity data. The CometChip® assay was developed to assess DNA strand breaks in a higher-throughput format. We paired the TGx-DDI biomarker with the CometChip® assay in TK6 cells to evaluate three model agents: nitrofurantoin (NIT), metronidazole (MTZ), and novobiocin (NOV). TGx-DDI was analyzed by two independent labs and technologies (nCounter® and TempO-Seq®). Although these anti-infective drugs are, or have been, used in human and/or veterinary medicine, the standard genotoxicity testing battery showed significant genetic safety findings. Specifically, NIT is a mutagen and causes chromosome damage, and MTZ and NOV cause chromosome damage in conventional in vitro tests. Herein, the TGx-DDI biomarker classified NIT and MTZ as non-DDI at all concentrations tested, suggesting that NIT's mutagenic activity is bacterial specific and that the observed chromosome damage by MTZ might be a consequence of in vitro test conditions. In contrast, NOV was classified as DDI at the second highest concentration tested, which is in line with the fact that NOV is a bacterial DNA-gyrase inhibitor that also affects topoisomerase II at high concentrations. The lack of DNA damage for NIT and MTZ was confirmed by the CometChip® results, which were negative for all three drugs except at overtly cytotoxic concentrations. This case study demonstrates the utility of combining the TGx-DDI biomarker and CometChip® to resolve conflicting genotoxicity data and provides further validation to support the reproducibility of the biomarker.

Oral N-acetylcysteine decreases IFN-γ production and ameliorates ischemia-reperfusion injury in steatotic livers.

Type 1 Natural Killer T-cells (NKT1 cells) play a critical role in mediating hepatic ischemia-reperfusion injury (IRI). Although hepatic steatosis is a major risk factor for preservation type injury, how NKT cells impact this is understudied. Given NKT1 cell activation by phospholipid ligands recognized presented by CD1d, we hypothesized that NKT1 cells are key modulators of hepatic IRI because of the increased frequency of activating ligands in the setting of hepatic steatosis. We first demonstrate that IRI is exacerbated by a high-fat diet (HFD) in experimental murine models of warm partial ischemia. This is evident in the evaluation of ALT levels and Phasor-Fluorescence Lifetime (Phasor-FLIM) Imaging for glycolytic stress. Polychromatic flow cytometry identified pronounced increases in CD45+CD3+NK1.1+NKT1 cells in HFD fed mice when compared to mice fed a normal diet (ND). This observation is further extended to IRI, measuring ex vivo cytokine expression in the HFD and ND. Much higher interferon-gamma (IFN-γ) expression is noted in the HFD mice after IRI. We further tested our hypothesis by performing a lipidomic analysis of hepatic tissue and compared this to Phasor-FLIM imaging using "long lifetime species", a byproduct of lipid oxidation. There are higher levels of triacylglycerols and phospholipids in HFD mice. Since N-acetylcysteine (NAC) is able to limit hepatic steatosis, we tested how oral NAC supplementation in HFD mice impacted IRI. Interestingly, oral NAC supplementation in HFD mice results in improved hepatic enhancement using contrast-enhanced magnetic resonance imaging (MRI) compared to HFD control mice and normalization of glycolysis demonstrated by Phasor-FLIM imaging. This correlated with improved biochemical serum levels and a decrease in IFN-γ expression at a tissue level and from CD45+CD3+CD1d+ cells. Lipidomic evaluation of tissue in the HFD+NAC mice demonstrated a drastic decrease in triacylglycerol, suggesting downregulation of the PPAR-γ pathway.

3,3'-Diindolylmethane Enhances Tumor Regression After Radiation Through Protecting Normal Cells to Modulate Antitumor Immunity.

PURPOSE: Preclinical and clinical data indicate that radiation therapy acts as an immune modifier, having both immune-stimulatory and immunosuppressive effects on the tumor-immune microenvironment (TIME). 3.3'-diindolylmethane (DIM) sensitizes tumor cells to radiation and protects mice from lethal doses of total body irradiation. We hypothesize that protecting nontumoral cells from the adverse effects of radiation treatment (RT) may help to correct immunosuppression resulting from radiation. METHODS AND MATERIALS: We generated tumor graft models using immune-competent and immune-deficient mouse strains. Narrow-beamed radiation was targeted to tumor sites using shielding. Tumor regression was monitored after DIM and RT versus RT alone. The effects of DIM on the efficacy of RT were assessed using immunohistochemistry staining and gene expression profiling. Complete blood counts, clonogenic cell survival assays, and global gene expression profiling of cultured cells were performed to study DIM's radioprotective effects on normal cells. RESULTS: DIM enhanced tumor regression after RT in immune-competent but not immune-deficient mice. Data indicated that DIM increased intratumoral immune cells after RT, contributing to enhanced immunologic responses such as adhesion and antigen processing. DIM protected normal cells from radiation-induced immediate injuries in vitro and in vivo. Transcriptomic profiling of cultured cells showed that DIM treatment mildly increased expression of some genes that are normally induced after radiation, such as genes involved in cell cycle arrest and apoptosis. CONCLUSIONS: In this study, using cultured cells and preclinical breast cancer models, we show that DIM protects normal cells from radiation-induced immediate cellular injury and combination treatment of DIM and radiation potentiates antitumor immune responses and enhances the efficacy of RT.

Radiochemotherapy upregulates expression of checkpoint receptors on circulating T cells.

PURPOSE: This study assesses changes of circulating leukocyte subpopulations and the expression of checkpoint receptors in T cells in patients undergoing radiochemotherapy. MATERIALS AND METHODS: Fifty-seven patients with either esophageal cancer or cervical cancer who received radiochemotherapy were recruited into this study. Serial blood collection was carried out before and during treatments. Leukocyte subpopulations and the level of PD-1 and CTLA-4 in T cells were determined by flow cytometry. The plasma concentrations of 34 human cytokines, chemokines, and growth factors were quantified. RESULTS: Significant decreases of lymphocyte count and percentage of T cells and B cells were observed during radiochemotherapy. Percentages of PD-1hi and CTLA-4hi populations in T cells increased after treatments. Proportion of activated T cells showed no significant difference. No significant changes in the plasma concentrations of the 34 humoral mediators except mild decreases of six cytokines. CONCLUSION: Elevated expression of PD-1 and CTLA-4 in T cells in patients receiving radiochemotherapy, which suggests that exhaustion-like T-cell dysfunction develops during cancer cytotoxic treatments.

Serum Metabolomic Alterations Associated with Cesium-137 Internal Emitter Delivered in Various Dose Rates.

Our laboratory and others have use radiation metabolomics to assess responses in order to develop biomarkers reflecting exposure and level of injury. To expand the types of exposure and compare to previously published results, metabolomic analysis has been carried out using serum samples from mice exposed to 137Cs internal emitters. Animals were injected intraperitoneally with 137CsCl solutions of varying radioactivity, and the absorbed doses were calculated. To determine the dose rate effect, serum samples were collected at 2, 3, 5, 7, and 14 days after injection. Based on the time for each group receiving the cumulative dose of 4 Gy, the dose rate for each group was determined. The dose rates analyzed were 0.16 Gy/day (low), 0.69 Gy/day (medium), and 1.25 Gy/day (high). The results indicated that at a cumulative dose of 4 Gy, the low dose rate group had the least number of statistically significantly differential spectral features. Some identified metabolites showed common changes for different dose rates. For example, significantly altered levels of oleamide and sphingosine 1-phosphate were seen in all three groups. On the other hand, the intensity of three amino acids, Isoleucine, Phenylalanine and Arginine, significantly decreased only in the medium dose rate group. These findings have the potential to be used in assessing the exposure and the biological effects of internal emitters.

Application of the adverse outcome pathway framework to genotoxic modes of action.

In May 2017, the Health and Environmental Sciences Institute's Genetic Toxicology Technical Committee hosted a workshop to discuss whether mode of action (MOA) investigation is enhanced through the application of the adverse outcome pathway (AOP) framework. As AOPs are a relatively new approach in genetic toxicology, this report describes how AOPs could be harnessed to advance MOA analysis of genotoxicity pathways using five example case studies. Each of these genetic toxicology AOPs proposed for further development includes the relevant molecular initiating events, key events, and adverse outcomes (AOs), identification and/or further development of the appropriate assays to link an agent to these events, and discussion regarding the biological plausibility of the proposed AOP. A key difference between these proposed genetic toxicology AOPs versus traditional AOPs is that the AO is a genetic toxicology endpoint of potential significance in risk characterization, in contrast to an adverse state of an organism or a population. The first two detailed case studies describe provisional AOPs for aurora kinase inhibition and tubulin binding, leading to the common AO of aneuploidy. The remaining three case studies highlight provisional AOPs that lead to chromosome breakage or mutation via indirect DNA interaction (inhibition of topoisomerase II, production of cellular reactive oxygen species, and inhibition of DNA synthesis). These case studies serve as starting points for genotoxicity AOPs that could ultimately be published and utilized by the broader toxicology community and illustrate the practical considerations and evidence required to formalize such AOPs so that they may be applied to genetic toxicity evaluation schemes. Environ. Mol. Mutagen. 61:114-134, 2020. © 2019 Wiley Periodicals, Inc.

TGx-DDI, a Transcriptomic Biomarker for Genotoxicity Hazard Assessment of Pharmaceuticals and Environmental Chemicals.

Genotoxicity testing is an essential component of the safety assessment paradigm required by regulatory agencies world-wide for analysis of drug candidates, and environmental and industrial chemicals. Current genotoxicity testing batteries feature a high incidence of irrelevant positive findings-particularly for in vitro chromosomal damage (CD) assays. The risk management of compounds with positive in vitro findings is a major challenge and requires complex, time consuming, and costly follow-up strategies including animal testing. Thus, regulators are urgently in need of new testing approaches to meet legislated mandates. Using machine learning, we identified a set of transcripts that responds predictably to DNA-damage in human cells that we refer to as the TGx-DDI biomarker, which was originally referred to as TGx-28.65. We proposed to use this biomarker in conjunction with current genotoxicity testing batteries to differentiate compounds with irrelevant "false" positive findings in the in vitro CD assays from true DNA damaging agents (i.e., for de-risking agents that are clastogenic in vitro but not in vivo). We validated the performance of the TGx-DDI biomarker to identify true DNA damaging agents, assessed intra- and inter- laboratory reproducibility, and cross-platform performance. Recently, to augment the application of this biomarker, we developed a high-throughput cell-based genotoxicity testing system using the NanoString nCounter® technology. Here, we review the status of TGx-DDI development, its integration in the genotoxicity testing paradigm, and progress to date in its qualification at the US Food and Drug Administration (FDA) as a drug development tool. If successfully validated and implemented, the TGx-DDI biomarker assay is expected to significantly augment the current strategy for the assessment of genotoxic hazards for drugs and chemicals.

Assessment of the performance of the TGx-DDI biomarker to detect DNA damage-inducing agents using quantitative RT-PCR in TK6 cells.

Gene expression biomarkers are now available for application in the identification of genotoxic hazards. The TGx-DDI transcriptomic biomarker can accurately distinguish DNA damage-inducing (DDI) from non-DDI exposures based on changes in the expression of 64 biomarker genes. The 64 genes were previously derived from whole transcriptome DNA microarray profiles of 28 reference agents (14 DDI and 14 non-DDI) after 4 h treatments of TK6 human lymphoblastoid cells. To broaden the applicability of TGx-DDI, we tested the biomarker using quantitative RT-PCR (qPCR), which is accessible to most molecular biology laboratories. First, we selectively profiled the expression of the 64 biomarker genes using TaqMan qPCR assays in 96-well arrays after exposing TK6 cells to the 28 reference agents for 4 h. To evaluate the classification capability of the qPCR profiles, we used the reference qPCR signature to classify 24 external validation chemicals using two different methods-a combination of three statistical analyses and an alternative, the Running Fisher test. The qPCR results for the reference set were comparable to the original microarray biomarker; 27 of the 28 reference agents (96%) were accurately classified. Moreover, the two classification approaches supported the conservation of TGx-DDI classification capability using qPCR; the combination of the two approaches accurately classified 21 of the 24 external validation chemicals, demonstrating 100% sensitivity, 81% specificity, and 91% balanced accuracy. This study demonstrates that qPCR can be used when applying the TGx-DDI biomarker and will improve the accessibility of TGx-DDI for genotoxicity screening. Environ. Mol. Mutagen. 60: 122-133, 2019. © 2018 Her Majesty the Queen in Right of Canada Environmental and Molecular Mutagenesis.

Prior irradiation results in elevated programmed cell death protein 1 (PD-1) in T cells.

PURPOSE: In this study we addressed the question whether radiation-induced adverse effects on T cell activation are associated with alterations of T cell checkpoint receptors. MATERIALS AND METHODS: Expression levels of checkpoint receptors on T cell subpopulations were analyzed at multiple post-radiation time points ranging from one to four weeks in mice receiving a single fraction of 1 or 4 Gy of γ-ray. T cell activation associated metabolic changes were assessed. RESULTS: Our results showed that prior irradiation resulted in significant elevated expression of programmed cell death protein 1 (PD-1) in both CD4+ and CD8+ populations, at all three post-radiation time points. T cells with elevated PD-1 mostly were either central memory or naïve cells. In addition, the feedback induction of PD-1 expression in activated T cells declined after radiation. CONCLUSION: Taken together, the elevated PD-1 level observed at weeks after radiation exposure is connected to T cell dysfunction. Recent preclinical and clinical studies have showed that a combination of radiotherapy and T cell checkpoint blockade immunotherapy including targeting the programmed death-ligand 1 (PD-L1)/PD-1 axis may potentiate the antitumor response. Understanding the dynamic changes in PD-1 levels in T cells after radiation should help in the development of a more effective therapeutic strategy.