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There is growing recognition across broad sectors of the scientific community that use of genomic biomarkers has the potential to reduce the need for conventional rodent carcinogenicity studies of industrial chemicals, agrochemicals, and pharmaceuticals through a weight-of-evidence approach. These biomarkers fall into 2 major categories: (1) sets of gene transcripts that can identify distinct tumorigenic mechanisms of action; and (2) cancer driver gene mutations indicative of rapidly expanding growth-advantaged clonal cell populations. This call-to-action article describes a collaborative approach launched to develop and qualify biomarker gene expression panels that measure widely accepted molecular pathways linked to tumorigenesis and their activation levels to predict tumorigenic doses of chemicals from short-term exposures. Growing evidence suggests that application of such biomarker panels in short-term exposure rodent studies can identify both tumorigenic hazard and tumorigenic activation levels for chemical-induced carcinogenicity. In the future, this approach will be expanded to include methodologies examining mutations in key cancer driver gene mutation hotspots as biomarkers of both genotoxic and nongenotoxic chemical tumor risk. Analytical, technical, and biological validation studies of these complementary genomic tools are being undertaken by multisector and multidisciplinary collaborative teams within the Health and Environmental Sciences Institute. Success from these efforts will facilitate the transition from current heavy reliance on conventional 2-year rodent carcinogenicity studies to more rapid animal- and resource-sparing approaches for mechanism-based carcinogenicity evaluation supporting internal and regulatory decision-making.

Supplementary Figure 1 from Carcinogen-Specific Gene Expression Profiles in Short-term Treated Eker and Wild-type Rats Indicative of Pathways Involved in Renal Tumorigenesis

Supplementary Table 4 from Carcinogen-Specific Gene Expression Profiles in Short-term Treated Eker and Wild-type Rats Indicative of Pathways Involved in Renal Tumorigenesis

Eker rats heterozygous for a dominant germline mutation in the tuberous sclerosis 2 (Tsc2) tumor suppressor gene were used as a model to study renal carcinogenesis. Eker and corresponding wild-type rats were exposed to genotoxic aristolochic acid (AA) or non-genotoxic ochratoxin A (OTA) to elucidate early carcinogen-specific gene expression changes and to test whether Eker rats are more sensitive to carcinogen-induced changes in gene expression. Male Eker and wild-type rats were gavaged daily with AA (10 mg/kg body weight) or OTA (210 μg/kg body weight). After 1, 3, 7, and 14 days of exposure, renal histopathology, tubular cell proliferation, and Affymetrix gene expression profiles from renal cortex/outer medulla were analyzed. AA-treated Eker and wild-type rats were qualitatively comparable in all variables assessed, suggesting a Tsc2-independent mechanism of action. OTA treatment resulted in slightly increased cortical pathology and significantly elevated cell proliferation in both strains, although Eker rats were more sensitive. Deregulated genes involved in the phosphatidylinositol 3-kinase-AKT-Tsc2-mammalian target of rapamycin signaling, among other important genes prominent in tumorigenesis, in conjunction with the enhanced cell proliferation and presence of preneoplastic lesions suggested involvement of Tsc2 in OTA-mediated toxicity and carcinogenicity, especially as deregulation of genes involved in this pathway was more prominent in the Tsc2 mutant Eker rat. [Cancer Res 2007;67(9):4052–68]

Supplementary Table 2 from Carcinogen-Specific Gene Expression Profiles in Short-term Treated Eker and Wild-type Rats Indicative of Pathways Involved in Renal Tumorigenesis

Supplementary Table 1 from Carcinogen-Specific Gene Expression Profiles in Short-term Treated Eker and Wild-type Rats Indicative of Pathways Involved in Renal Tumorigenesis

Supplementary Table 3 from Carcinogen-Specific Gene Expression Profiles in Short-term Treated Eker and Wild-type Rats Indicative of Pathways Involved in Renal Tumorigenesis

Drug-induced kidney injury (DIKI) is a major concern in both drug development and clinical practice. There is an unmet need for biomarkers of glomerular damage and more distal renal injury in the loop of Henle and the collecting duct (CD). A cross-laboratory program to identify and characterize urinary microRNA (miRNA) patterns reflecting tissue- or pathology-specific DIKI was conducted. The overall goal was to propose miRNA biomarker candidates for DIKI that could supplement information provided by protein kidney biomarkers in urine. Rats were treated with nephrotoxicants causing injury to distinct nephron segments: the glomerulus, proximal tubule, thick ascending limb (TAL) of the loop of Henle and CD. Meta-analysis identified miR-192-5p as a potential proximal tubule-specific urinary miRNA candidate. This result was supported by data obtained in laser capture microdissection nephron segments showing that miR-192-5p expression was enriched in the proximal tubule. Discriminative miRNAs including miR-221-3p and -222-3p were increased in urine from rats treated with TAL versus proximal tubule toxicants in accordance with their expression localization in the kidney. Urinary miR-210-3p increased up to 40-fold upon treatment with TAL toxicants and was also enriched in laser capture microdissection samples containing TAL and/or CD versus proximal tubule. miR-23a-3p was enriched in the glomerulus and was increased in urine from rats treated with doxorubicin, a glomerular toxicant, but not with toxicants affecting other nephron segments. Taken together these results suggest that urinary miRNA panels sourced from specific nephron regions may be useful to discriminate the pathology of toxicant-induced lesions in the kidney, thereby contributing to DIKI biomarker development needs for industry, clinical, and regulatory use.

Micro-RNAs (miRNAs) are regulators of gene expression and play an important role in physiological homeostasis and disease. In biofluids, miRNAs can be found in protein complexes or in extracellular vesicles (EVs). Altered urinary miRNAs are reported as potential biomarkers for chronic kidney disease (CKD). In this context, we compared established urinary protein biomarkers for kidney injury with urinary miRNA profiles in obese ZSF1 and hypertensive renin transgenic rats. Additionally, the benefit of urinary EV enrichment was investigated in vivo and the potential association of urinary miRNAs with renal fibrosis in vitro. Kidney damage in both rat models was confirmed by histopathology, proteinuria, and increased levels of urinary protein biomarkers. In total, 290 miRNAs were elevated in obese ZSF1 rats compared with lean controls, whereas 38 miRNAs were altered in obese ZSF1 rats during 14–26 weeks of age. These 38 miRNAs correlated better with disease progression than established urinary protein biomarkers. MiRNAs increased in obese ZSF1 rats were associated with renal inflammation, fibrosis, and glomerular injury. Eight miRNAs were also changed in urinary EVs of renin transgenic rats, including one which might play a role in endothelial dysfunction. EV enrichment increased the number and detection level of several miRNAs implicated in renal fibrosis in vitro and in vivo. Our results show the benefit of EV enrichment for miRNA detection and the potential of total urine and urinary EV-associated miRNAs as biomarkers of altered kidney physiology, renal fibrosis and glomerular injury, and disease progression in hypertension and obesity-induced CKD.

Purpose Fibroblast growth factor receptor 2 (FGFR2) has been previously reported to be overexpressed in several types of cancer, whereas the expression in normal tissue is considered to be moderate to low. Thus, FGFR2 is regarded as an attractive tumor antigen for targeted alpha therapy. This study reports the evaluation of an FGFR2-targeted thorium-227 conjugate (FGFR2-TTC, BAY 2304058) comprising an anti-FGFR2 antibody, a chelator moiety covalently conjugated to the antibody, and the alpha particle–emitting radionuclide thorium-227. FGFR2-TTC was assessed as a monotherapy and in combination with the DNA damage response inhibitor ATRi BAY 1895344. Methods and Materials The in vitro cytotoxicity and mechanism of action were evaluated by determining cell viability, the DNA damage response marker γH2A.X, and cell cycle analyses. The in vivo efficacy was determined using human tumor xenograft models in nude mice. Results In vitro mechanistic assays demonstrated upregulation of γH2A.X and induction of cell cycle arrest in several FGFR2-expressing cancer cell lines after treatment with FGFR2-TTC. In vivo, FGFR2-TTC significantly inhibited tumor growth at a dose of 500 kBq/kg in the xenograft models NCI-H716, SNU-16, and MFM-223. By combining FGFR2-TTC with the ATR inhibitor BAY 1895344, an increased potency was observed in vitro, as were elevated levels of γH2A.X and inhibition of FGFR2-TTC–mediated cell cycle arrest. In the MFM-223 tumor xenograft model, combination of the ATRi BAY 1895344 with FGFR2-TTC resulted in significant tumor growth inhibition at doses at which the single agents had no effect. Conclusions The data provide a mechanism-based rationale for combining the FGFR2-TTC with the ATRi BAY 1895344 as a new therapeutic approach for treatment of FGFR2-positive tumors from different cancer indications.

The biguanide metformin, a widely used antidiabetic drug, has received great interest in oncology research in recent years after an epidemiological study showed a link between metformin treatment and a reduced cancer risk in diabetic patients. Since mitochondrial metabolism has become a target for possible cancer therapeutic approaches, especially for tumors relying on oxidative metabolism, mitochondrial complex I inhibition is under discussion to be responsible for the anti-cancer effect of metformin. Rotenone, a well-known strong mitochondrial complex I inhibitor, yet associated with toxic effects, has also shown anti-cancer activity. Thus, we compared metformin and phenformin, another biguanide previously on the market as antidiabetic, with rotenone, to elucidate potential mechanisms rendering biguanides apparently less toxic than rotenone. Therefore, we conducted in vivo rat studies with metformin and phenformin, based on an experimental design previously described for mechanistic investigations of the effects of rotenone, including blood and tissue analysis, histopathology and gene expression profiling. These investigations show that the mechanistic profile of phenformin appears similar to that of rotenone, yet at a quantitatively reduced level, whereas metformin displays only transient similarities after one day of treatment. A potential reason may be that metformin, but not rotenone or phenformin, self-limits its entry into mitochondria due to its molecular properties. Thus, our detailed molecular characterization of these compounds suggests that inhibition of mitochondrial functions can serve as target for an anti-cancer mode of action, but should be self-limited or balanced to some extent to avoid exhaustion of all energy stores.

Drug-induced liver injury (DILI) cannot be accurately predicted by animal models. In addition, currently available in vitro methods do not allow for the estimation of hepatotoxic doses or the determination of an acceptable daily intake (ADI). To overcome this limitation, an in vitro/in silico method was established that predicts the risk of human DILI in relation to oral doses and blood concentrations. This method can be used to estimate DILI risk if the maximal blood concentration (Cmax) of the test compound is known. Moreover, an ADI can be estimated even for compounds without information on blood concentrations. To systematically optimize the in vitro system, two novel test performance metrics were introduced, the toxicity separation index (TSI) which quantifies how well a test differentiates between hepatotoxic and non-hepatotoxic compounds, and the toxicity estimation index (TEI) which measures how well hepatotoxic blood concentrations in vivo can be estimated. In vitro test performance was optimized for a training set of 28 compounds, based on TSI and TEI, demonstrating that (1) concentrations where cytotoxicity first becomes evident in vitro (EC10) yielded better metrics than higher toxicity thresholds (EC50); (2) compound incubation for 48 h was better than 24 h, with no further improvement of TSI after 7 days incubation; (3) metrics were moderately improved by adding gene expression to the test battery; (4) evaluation of harmacokinetic parameters demonstrated that total blood compound concentrations and the 95%-population-based percentile of Cmax were best suited to estimate human toxicity. With a support vector machine-based classifier, using EC10 and Cmax as variables, the cross-validated sensitivity, specificity and accuracy for hepatotoxicity prediction were 100, 88 and 93%, respectively. Concentrations in the culture medium allowed extrapolation to blood concentrations in vivo that are associated with a specific probability of hepatotoxicity and the corresponding oral doses were obtained by reverse modeling. Application of this in vitro/in silico method to the rat hepatotoxicant pulegone resulted in an ADI that was similar to values previously established based on animal experiments. In conclusion, the proposed method links oral doses and blood concentrations of test compounds to the probability of hepatotoxicity.

MicroRNAs (miRNAs) are small non-coding RNA that regulate the expression of messenger RNA and are implicated in almost all cellular processes. Importantly, miRNAs are can be released extracellularly and are stable in these matrices where they may serve as indicators of organ or cell-specific toxicity, disease, and biological status. There has thus been great enthusiasm for developing miRNAs as biomarkers of adverse outcomes for scientific, regulatory, and clinical purposes. Despite advances in measurement capabilities for miRNAs, miRNAs are still not routinely employed as non-invasive biomarkers. This is in part due to the lack of standard approaches for sample preparation and miRNA measurement and uncertainty in their biological interpretation. Members of the microRNA Biomarkers Workgroup within the Health and Environmental Sciences Institute’s (HESI) Committee on Emerging Systems Toxicology for the Assessment of Risk (eSTAR) are a consortium of private- and public-sector scientists dedicated to developing miRNAs as applied biomarkers. Here, we explore major impediments to routine acceptance and use of miRNA biomarkers and case examples of success and deficiencies in development. Finally, we provide insight on miRNA measurement, collection, and analysis tools to provide solid footing for addressing knowledge gaps toward routine biomarker use.

A cross-industry survey was conducted by EFPIA/IQ DruSafe in 2018 to provide information on photosafety evaluation of pharmaceuticals after implementation of ICH S10. This survey focused on the strategy utilized for photosafety risk assessment, the design of nonclinical (in vitro and in vivo) and clinical evaluations, the use of exposure margins in risk assessment, and regulatory interactions. The survey results indicated that a staged approach for phototoxicity assessment has been widely accepted by regulatory authorities globally. The OECD-based 3T3 NRU Phototoxicity Test is the most frequently used in vitro approach. Modifications to this assay suggested by ICH S10 are commonly applied. For in-vitro-positives, substantial margins from in vitro IC50 values under irradiation to Cmax (clinical) have enabled further development without the need for additional photosafety data. In vivo phototoxicity studies typically involve dosing rodents and exposing skin and eyes to simulated sunlight, and subsequently evaluating at least the skin for erythema and edema. However, no formal guidelines exist and protocols are less standardized across companies. A margin-of-safety approach (based on Cmax at NOAEL) has been successfully applied to support clinical development. Experience with dedicated clinical phototoxicity studies was limited, perhaps due to effective de-risking approaches employed based on ICH S10.

Recently, bile acids (BAs) were reported as promising markers for drug-induced liver injury (DILI). BAs have been suggested to correlate with hepatocellular and hepatobiliary damage; however a clear connection of BA patterns with different types of DILI remains to be established. To investigate if BAs can improve the assessment of liver injury, 20 specific BAs were quantitatively profiled via LC-MS/MS in plasma and liver tissue in a model of methapyrilene-induced liver injury in rats. Methapyrilene, a known hepatotoxin was dosed daily over 14-days at doses of 30 and 80mg/kg, followed by a recovery phase of 10days. Conventional preclinical safety endpoints were related to BA perturbations and to hepatic gene expression profiling for a mechanistic interpretation of effects. Histopathological signs of hepatocellular and hepatobiliary damage with significant changes of clinical chemistry markers were accompanied by significantly increased levels of indivdual BAs in plasma and liver tissue. BA perturbations were already evident at the earliest time point after 30mg/kg treatment, and thereby indicating better sensitivity than clinical chemistry parameters. Furthermore, the latter markers suggested recovery of liver injury, whereas BA levels in plasma and liver remained significantly elevated during the recovery phase, in line with persistent histopathological findings of bile duct hyperplasia (BDH) and bile pigment deposition. Gene expression profiling revealed downregulation of genes involved in BA synthesis (AMACR, BAAT, ACOX2) and hepatocellular uptake (NTCP, OATs), and upregulation for efflux transporters (MRP2, MRP4), suggesting an adaptive hepatocellular protection mechanism against cytotoxic bile acid accumulation. In summary, our data suggests that specific BAs with high reliability such as cholic acid (CA) and chenodeoxycholic acid (CDCA) followed by glycocholic acid (GCA), taurocholic acid (TCA) and deoxycholic acid (DCA) can serve as additional biomarkers for hepatocellular/hepatobiliary damage in the liver in rat toxicity studies.

Derisking xenobiotic-induced nongenotoxic carcinogenesis (NGC) represents a significant challenge during the safety assessment of chemicals and therapeutic drugs. The identification of robust mechanism-based NGC biomarkers has the potential to enhance cancer hazard identification. We previously demonstrated Constitutive Androstane Receptor (CAR) and WNT signaling-dependent up-regulation of the pluripotency associated Dlk1-Dio3 imprinted gene cluster noncoding RNAs (ncRNAs) in the liver of mice treated with tumor-promoting doses of phenobarbital (PB). Here, we have compared phenotypic, transcriptional ,and proteomic data from wild-type, CAR/PXR double knock-out and CAR/PXR double humanized mice treated with either PB or chlordane, and show that hepatic Dlk1-Dio3 locus long ncRNAs are upregulated in a CAR/PXR-dependent manner by two structurally distinct CAR activators. We further explored the specificity of Dlk1-Dio3 locus ncRNAs as hepatic NGC biomarkers in mice treated with additional compounds working through distinct NGC modes of action. We propose that up-regulation of Dlk1-Dio3 cluster ncRNAs can serve as an early biomarker for CAR activator-induced nongenotoxic hepatocarcinogenesis and thus may contribute to mechanism-based assessments of carcinogenicity risk for chemicals and novel therapeutics.

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.

Background Circulating microRNAs are undergoing exploratory use as safety biomarkers in drug development. Reverse transcription quantitative polymerase chain reaction (RT-qPCR) is one common approach used to quantitate levels of microRNAs in samples that includes the use of a standard curve of calibrators fit to a regression model. Guidelines are needed for setting assay quantitation thresholds that are appropriate for this method and to biomarker pre-validation. Results In this report, we develop two workflows for determining a lower limit of quantitation (LLOQ) for RT-qPCR assays of microRNAs in exploratory studies. One workflow is based on an error threshold calculated by a logistic model of the calibration curve data. The second workflow is based on a threshold set by the sample blank, which is the no template control for RT-qPCR. The two workflows are used to set lower thresholds of reportable microRNA levels for an example dataset in which miR-208a levels in biofluids are quantitated in a cardiac injury model. LLOQ thresholds set by either workflow are effective in filtering out microRNA values with large uncertainty estimates. Conclusions Two workflows for LLOQ determinations are presented in this report that provide methods that are easy to implement in investigational studies of microRNA safety biomarkers and offer choices in levels of conservatism in setting lower limits of acceptable values that facilitate interpretation of results. Electronic supplementary material The online version of this article (10.1186/s12896-018-0415-4) contains supplementary material, which is available to authorized users.

Identification of circulating microRNAs for the diagnosis of liver injury and as an indicator of underlying pathology has been the subject of recent investigations. While several studies have been conducted, with particular emphasis on miR-122, the timing of miRNA release into the circulation and anchoring to tissue pathology has not been systematically evaluated. In this study, miRNA profiling was conducted over a time course of hepatobiliary injury and repair using alpha-naphthylisothiocyanate (ANIT) and a proprietary compound, FP004BA. ANIT administration (50 mg/kg) to rats caused significant biliary epithelial cell and hepatocellular necrosis between 24 and 72 h, followed by resolution and progression to biliary hyperplasia by 120 h which was associated with miRNA release into the blood. FP004BA (100 mg/kg) was used to confirm associations of miRNA along a time course with similar hepatic pathology to ANIT. Treatment with ANIT or FP004BA resulted in significant alterations of overlapping miRNAs during the early and peak injury phases. In addition to well-characterized liver injury markers miR-122‐5p and miR-192‐5p, multiple members of the 200 family and the 101 family along with miR-802‐5p and miR-30d‐5p were consistently elevated during hepatobiliary injury caused by both toxicants, suggesting that these species may be potential biomarker candidates for hepatobiliary injury. After 14 days of dosing with 4BA, miR-182‐5p remained elevated—while miR-122‐5p and miR-192‐5p had returned to baseline—suggesting that miR-182‐5p may have added utility to monitor for hepatobiliary injury in the repair phases when there remains histological evidence of ongoing cellular injury.

MicroRNAs (miRNAs) regulate gene expression post-transcriptionally and thus are involved in various physiological and pathological states. Due to their stability in biofluids miRNAs have also been proposed as biomarkers (BMs) for tissue injury. We investigated the usefulness of urinary miRNAs for detection of site-specific renal damage in an antiglomerular basement membrane glomerulonephritis (GN) model in rats by comparing GN-induced urinary miRNAs profiles to traditional and newer protein BMs, and to proximal tubular injury-induced urinary miRNA profiles observed previously after cisplatin (Cp) treatment. Male Wistar Kyoto and Sprague Dawley rats were dosed once with 1, 2.5, and 5 ml/kg nephrotoxic serum (NTS) or 1.5 and 5 ml/kg NTS, respectively. GN and tubular damage were observed histopathologically in all treated rats after 14 days. Although serum creatinine and BUN were not changed, several protein BMs and 74 urinary miRNAs were found to be increased 8 and 14 days after NTS administration. Of these 74 miRNAs, 5 were identified as increased after NTS but not after Cp treatment. Using in situ hybridization two of them, miR-10 b and -100, were found to be localized in distal segments of the nephron, potentially reflecting the tubular injury in those regions. Furthermore, evaluation of both miRNA and mRNA expression in the kidney revealed possible miRNA-mRNA interactions mostly associated with fibrotic and transforming growth factor β signaling. In conclusion, our investigations support the potential of urinary miRNAs as specific BMs for kidney injury, and suggest a role of miRNAs in pathological processes during GN in the kidney.

A major reason for the current reproducibility crisis in the life sciences is the poor implementation of quality control measures and reporting standards. Improvement is needed, especially regarding increasingly complex in vitro methods. Good Cell Culture Practice (GCCP) was an effort from 1996 to 2005 to develop such minimum quality standards also applicable in academia. This paper summarizes recent key developments in in vitro cell culture and addresses the issues resulting for GCCP, e.g. the development of induced pluripotent stem cells (iPSCs) and gene-edited cells. It further deals with human stem-cell-derived models and bioengineering of organo-typic cell cultures, including organoids, organ-on-chip and human-on-chip approaches. Commercial vendors and cell banks have made human primary cells more widely available over the last decade, increasing their use, but also requiring specific guidance as to GCCP. The characterization of cell culture systems including high-content imaging and high-throughput measurement technologies increasingly combined with more complex cell and tissue cultures represent a further challenge for GCCP. The increasing use of gene editing techniques to generate and modify in vitro culture models also requires discussion of its impact on GCCP. International (often varying) legislations and market forces originating from the commercialization of cell and tissue products and technologies are further impacting on the need for the use of GCCP. This report summarizes the recommendations of the second of two workshops, held in Germany in December 2015, aiming map the challenge and organize the process or developing a revised GCCP 2.0. published

Transcriptomics is developing into an invaluable tool in toxicology. The aim of this study was, using a transcriptomics approach, to identify genes that respond similar to many different chemicals (including drugs and industrial compounds) in both rat liver in vivo and in cultivated hepatocytes. For this purpose, we analyzed Affymetrix microarray expression data from 162 compounds that were previously tested in a concentration-dependent manner in rat livers in vivo and in rat hepatocytes cultivated in sandwich culture. These data were obtained from the Japanese Toxicogenomics Project (TGP) and North Rhine-Westphalian (NRW) data sets, which represent 138 and 29 compounds, respectively, and have only 5 compounds in common between them. The in vitro gene expression data from the NRW data set were generated in the present study, while TGP is publicly available. For each of the data sets, the overlap between up- or down-regulated genes in vitro and in vivo was identified, and named in vitro-in vivo consensus genes. Interestingly, the in vivo-in vitro consensus genes overlapped to a remarkable extent between both data sets, and were 21-times (upregulated genes) or 12-times (down-regulated genes) enriched compared to random expectation. Finally, the genes in the TGP and NRW overlap were used to identify the upregulated genes with the highest compound coverage, resulting in a seven-gene set of Cyp1a1, Ugt2b1, Cdkn1a, Mdm2, Aldh1a1, Cyp4a3, and Ehhadh. This seven-gene set was then successfully tested with structural analogues of valproic acid that are not present in the TGP and NRW data sets. In conclusion, the seven-gene set identified in the present study responds similarly in vitro and in vivo to a wide range of different chemicals. Despite these promising results with the seven-gene set, transcriptomics with cultivated rat hepatocytes remains a challenge, because in general many genes are up- or downregulated by in vitro culture per se, respond differently to test compounds in vitro and in vivo, and/or show higher variability in the in vitro system compared to the corresponding in vivo data. published

Significance Standard in vitro assays to assess genotoxicity frequently generate positive results that are subsequently found to be irrelevant for in vivo carcinogenesis and human cancer risk assessment. Currently used follow-up methods, such as animal testing, are expensive and time-consuming, and the development of approaches enabling more accurate mechanism-based risk assessment is essential. We developed an in vitro transcriptomic biomarker-based approach that provides a robust biomarker reflecting stress-signaling responses. The biomarker correctly identifies the vast majority of irrelevant genotoxicity results from in vitro chromosome damage assays. TGx-DDI, a multigene biomarker for DNA damage-inducing agents, is the first biomarker that not only shows convincing interlaboratory and intralaboratory reproducibility, but also performs accurately in a system suitable for high-throughput screening., 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.

Inhibitors of the mitochondrial respiratory chain complex I are suggested to exert anti-tumor activity on those tumors relying on oxidative metabolism and are therefore of interest to oncology research. Nevertheless, the safety profile of these inhibitors should be thoroughly assessed. Rotenone, a proven complex I inhibitor, has shown anti-carcinogenic activity in several studies. In this context rotenone was used in this study as a tool compound with the aim to identify suitable biomarker candidates and provide enhanced mechanistic insights into the molecular and cellular effects of complex I inhibitors. Rats were treated with 400 ppm rotenone daily for 1, 3 or 14 consecutive days followed by necropsy. Classical clinical endpoints, including hematology, clinical chemistry and histopathology with supporting investigations (FACS-analysis, enzymatic activity assays) were examined as well as gene expression analysis. Through these investigations, we identified liver, bone marrow and bone as target organs amongst approx. 40 organs evaluated at least histopathologically. Our results suggest blood analysis, bone marrow parameters, assessment of lactate in serum and glycogen in liver, and especially gene expression analysis in liver as useful parameters for an experimental model to help to characterize the profile of complex I inhibitors with respect to a tolerable risk-benefit balance.

Inhibition of the glucagon receptor (GCGR) has been identified as a potential therapeutic approach for the treatment of type 2 diabetes. However, a small molecule drug candidate antagonizing GCGR (BAY16) failed during preclinical drug development, in part due to drug induced hepatotoxicity in animals. Since there is evidence to suggest that endogenous GCGR signaling might be important for hepatocyte survival, we hypothesized that on-target effects, i.e., modulation of GCGR activity by BAY16, may contribute to BAY16 hepatotoxicity and associated gene expression changes in rats. To understand the role of GCGR inhibition in BAY16 toxicity, we analyzed cell viability and gene expression profiles in non-silenced and GCGR-targeting siRNA transfected primary rat hepatocytes with and without exposure to BAY16 to discriminate between on- and off-target effects of BAY16. siRNA-mediated silencing of the GCGR did not affect cell viability in primary rat hepatocytes, indicating that cytotoxicity of BAY16 occurs independent of its pharmacological effects. In support of this, gene expression analysis of GCGR silenced hepatocytes revealed no transcriptional alterations relevant to toxicity. In contrast, BAY16 caused a concentration-dependent decrease in cell viability, along with changes in the expression of genes associated with altered xenobiotic metabolism, oxidative stress, increased fatty acid synthesis, and alterations in cholesterol and bile acid metabolic processes. Based on gene expression data, it appears that hepatocytes inhibit cholesterol synthesis and increase detoxifying and eliminating processes in order to protect themselves from accumulation of bile acids, cholesterol or drug intermediates. Importantly, comparison of transcriptional changes in the absence and presence of GCGR revealed that the same pathways were affected in both silenced and non-silenced hepatocytes, indicating that BAY16 toxicity occurs independent of the GCGR receptor.

The constitutive androstane receptor (CAR) and the pregnane X receptor (PXR) are closely related nuclear receptors involved in drug metabolism and play important roles in the mechanism of phenobarbital (PB)-induced rodent nongenotoxic hepatocarcinogenesis. Here, we have used a humanized CAR/PXR mouse model to examine potential species differences in receptor-dependent mechanisms underlying liver tissue molecular responses to PB. Early and late transcriptomic responses to sustained PB exposure were investigated in liver tissue from double knock-out CAR and PXR (CAR(KO)-PXR(KO)), double humanized CAR and PXR (CAR(h)-PXR(h)), and wild-type C57BL/6 mice. Wild-type and CAR(h)-PXR(h) mouse livers exhibited temporally and quantitatively similar transcriptional responses during 91 days of PB exposure including the sustained induction of the xenobiotic response gene Cyp2b10, the Wnt signaling inhibitor Wisp1, and noncoding RNA biomarkers from the Dlk1-Dio3 locus. Transient induction of DNA replication (Hells, Mcm6, and Esco2) and mitotic genes (Ccnb2, Cdc20, and Cdk1) and the proliferation-related nuclear antigen Mki67 were observed with peak expression occurring between 1 and 7 days PB exposure. All these transcriptional responses were absent in CAR(KO)-PXR(KO) mouse livers and largely reversible in wild-type and CAR(h)-PXR(h) mouse livers following 91 days of PB exposure and a subsequent 4-week recovery period. Furthermore, PB-mediated upregulation of the noncoding RNA Meg3, which has recently been associated with cellular pluripotency, exhibited a similar dose response and perivenous hepatocyte-specific localization in both wild-type and CAR(h)-PXR(h) mice. Thus, mouse livers coexpressing human CAR and PXR support both the xenobiotic metabolizing and the proliferative transcriptional responses following exposure to PB.

Introduction In the past years several new urinary nephrotoxicity biomarkers have been qualified for use in preclinical studies by the FDA and EMA. Subsequently, kits have been developed to measure these urinary biomarkers on multiplex platforms such as the electro-chemiluminescent based immunoassay from MesoScale Discovery (MSD) and the bead-based immunoassay using Luminex xMAP technology (LMX). The aim of the present study was to compare the two multiplex platforms with respect to the capability of their qualified urinary biomarker panels to measure an increase of these biomarkers relative to histopathological changes in an animal model of nephrotoxicity. Methods For comparison of the two platforms we used urine samples from a study with the well-characterized nephrotoxin cisplatin (Cp) in male Wistar rats. The following five biomarkers were measured on both platforms: glutathione S-transferase α (αGST), clusterin (CLU), kidney injury molecule-1 (KIM-1), neutrophil gelatinase-associated lipocalin (NGAL, a.k.a. lipocalin-2) and osteopontin (OPN). The measurements were compared with respect to both the fold increase observed for each biomarker and the absolute concentrations measured in relation to traditional endpoints for nephrotoxicity in clinical pathology and histopathology. Results The platform comparison revealed the expected increases of urinary biomarkers after Cp treatment with similar results at the fold change level enabling consistent detection of kidney injury. The comparison of the absolute concentrations of biomarkers measured in the two platforms showed differences, the extent of which was analyte-dependent. Discussion By comparison of two widely used multiplex platforms, MSD and LMX, for the detection of renal toxicity biomarkers in urine, we observed the expected increases of these biomarkers in response to Cp administration. Depending on the marker, significant differences could be found when comparing the absolute concentrations thus suggesting that baseline levels for each platform will have to be set separately.

Systemic inflammation co-activates coagulation, which unchecked culminates in a lethal syndrome of multi-organ microvascular thrombosis known as disseminated intravascular coagulation (DIC). We studied an endotoxin-induced inflammatory state in rats to identify biomarkers of hemostatic imbalance favoring hypercoagulability. Intraperitoneal injection of LPS at 15 mg/kg body weight resulted in peripheral leukopenia and widespread neutrophilic sequestration characteristic of an acute systemic inflammatory response. Early indicators of hemostatic pathway activation developed within 4 hours, including increased circulating concentrations of procoagulant extracellular vesicles (EVs), EVs expressing endothelial cell and platelet membrane markers, and high concentration of soluble intercellular adhesion molecule-1 (sICAM-1), plasminogen activator inhibitor-1 (PAI-1), and D-dimers. Inflammation persisted throughout the 48-hour observation period; however, increases were found in a subset of serum microRNA (miRNA) that coincided with gradual resolution of hemostatic protein abnormalities and reduction in EV counts. Dose-adjusted LPS treatment in rats provides a time-course model to develop biomarker profiles reflecting procoagulant imbalance and rebalance under inflammatory conditions.

Genotoxic carcinogens pose great hazard to human health. Uncertainty of current risk assessment strategies and long latency periods between first carcinogen exposure and diagnosis of tumors have raised interest in predictive biomarkers. Initial DNA adduct formation is a necessary step for genotoxin induced carcinogenesis. However, as DNA adducts not always translate into tumorigenesis, their predictive value is limited. Here we hypothesize that the combined analysis of pro-mutagenic DNA adducts along with time-matched gene expression changes could serve as a superior prediction tool for genotoxic carcinogenesis. Eker rats, heterozygous for the tuberous sclerosis (Tsc2) tumor suppressor gene and thus highly susceptible towards genotoxic renal carcinogens, were continuously treated with the DNA alkylating carcinogen methylazoxymethanol acetate (MAMAc). Two weeks of MAMAc treatment resulted in a time-dependent increase of O(6)-methylguanine and N7-methylguanine adducts in the kidney cortex, which was however not reflected by significant expression changes of cyto-protective genes involved in DNA repair, cell cycle arrest or apoptosis. Instead, we found a transcriptional regulation of genes involved in the tumor-related MAPK, FoxO and TGF-beta pathways. Continuous MAMAc treatment for up to 6 months resulted in a mild but significant increase of cancerous lesions. In summary, the combined analysis of DNA adducts and early gene expression changes could serve as a suitable predictive tool for genotoxicant-induced carcinogenesis. published

The late detection of non-genotoxic carcinogens in the drug development process can delay drug candidates for unmet medical needs from reaching the market despite considerable investments in their development. To enable faster, safer, and less expensive development of medications for patients, the MARCAR project generated a large set of transcriptomic data to investigate the underlying mechanisms of non-genotoxic hepatocarcinogenesis and to identify potential biomarkers for early detection of tumor formation in the rodent liver. The effective mining of these high-dimensional datasets is a non-trivial task that usually requires bioinformatics support to extract relevant mechanistic patterns and confirm toxicological hypotheses. Here, we present MARCARviz, a web-platform that enables biologists to (a) quickly address the most common questions associated with the MARCAR microarray data, to (b) identify relevant patterns in the data, and to (c) generate or confirm mechanistic hypotheses about non-genotoxic effects leading to cancer formation. The major advantage of MARCARviz is that there is no software or advanced technical knowledge required to perform powerful analyses and generate visualizations of the MARCAR data. MARCARviz greatly facilitates the confirmation of published MARCAR results and generation of new insights from the collected data by the greater public without the requirement for tedious pre-processing steps. MARCARviz is publicly available from https://tea.cs.uni-tuebingen.de/.

Cultivated hepatocytes represent a well-established in vitro system. However, the applicability of hepatocytes in toxicogenomics is still controversially discussed. Recently, an in vivo/in vitro discrepancy has been described, whereby the non-genotoxic rat liver carcinogen methapyrilene alters the expression of the metabolizing genes SULT1A1 and ABAT, as well as the DNA damage response gene GADD34 in vitro, but not in vivo. If the collagen sandwich cultures of hepatocytes really produce false-positive data, this would compromise its application in toxicogenomics. To revisit the putative in vivo/in vitro discrepancy, we first analyzed and modeled methapyrilene concentrations in the portal vein of rats. The relatively short half-life of 2.8 h implies a rapid decrease in orally administered methapyrilene in vivo below concentrations that can cause gene expression alterations. This corresponded to the time-dependent alteration levels of GADD34, ABAT and SULT1A1 RNA in the liver: RNA levels are altered 1, 6 and 12 h after methapyrilene administration, but return to control levels after 24 and 72 h. In contrast, methapyrilene concentrations in the culture medium supernatant of primary rat hepatocyte cultures decreased slowly. This explains why GADD34, ABAT and SULT1A1 were still deregulated after 24 h exposure in vitro, but not in vivo. It should also be considered that the earliest analyzed time point in the previous in vivo studies was 24 h after methapyrilene administration. In conclusion, previously observed in vitro/in vivo discrepancy can be explained by different pharmacokinetics present in vitro and in vivo. When the in vivo half-life is short, levels of some initially altered genes may have returned to control levels already 24 h after administration.

The concept of mechanistic toxicogenomics implies that compound-induced changes in gene expression profiles provide valuable information about their mode of action. A growing number of research groups have presented evidence that whole-genome gene expression profiling techniques might be used as tools for in vivo and in vitro generation of gene signatures and elucidation of molecular mechanisms after exposure to toxic compounds. An important issue to be investigated is the in vivo relevance of in vitro-obtained data. In the current study, we compare the gene expression profiles generated in vitro, after exposing conventional and epigenetically stabilized primary rat hepatocytes to well-known genotoxic hepatocarcinogens (aflatoxin B1, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone and 2-nitrofluorene) with those derived in vivo after oral exposure of rats to these compounds. Similar statistical tools were applied on both sets of data. The major molecular pathways affected in the in vivo setting were DNA damage, detoxification and cell survival response, as previously described. In the conventional hepatocyte cultures, two of the three genotoxicants showed quite similar responses as in vivo with respect to these pathways. The third compound (2-nitrofluorene) revealed in vitro response which was not observed in vivo. In the epigenetically stabilized hepatocytes, in contrast to what was expected, the responses were less relevant for the in vivo situation. This study highlights the importance of in vitro/in vivo comparison of data that are generated using in vitro models and shows that conventional primary rat hepatocyte cultures represent an appropriate in vitro model to retrieve mechanistic information on the exposure to genotoxicants.

Objective Nephrogenic systemic fibrosis (NSF) is an acquired, idiopathic disorder. Most of the cases are observed in patients with end stage renal disease (ESRD). The objective of this nonclinical animal study was to test the hypothesis that gadolinium (Gd) deposits play a role in the induction of NSF lesions. In addition, we evaluated whether an acute response to Gd exposure can initiate a process that results in fibrosis of the skin. Materials and methods Han-Wistar rats were administered 3 intravenous injections of Gd-DTPA-BMA formulated without Gd-free excess ligand (Gadodiamide without Caldiamide) at a dose of 2.5 mmol/kg of body weight (b.w.) per injection given at 24-hour or 14-, 28-, or 56-day intervals. The occurrence and development of NSF-like fibrosing dermopathy lesions were followed. The Gd concentration was determined by Inductively Coupled Plasma Mass Spectrometry in skin biopsies taken during the study and organ samples taken at the end of the study.In a separate study, after injection of a single intravenous dose of 2.5 mmol/kg b.w. Gd-DTPA-BMA administered to Han-Wistar rats, the expression of cytokines and signaling molecules in serum and skin tissue was determined by quantitative RT-PCR and Luminex technology 6 hours or 14, 28, or 56 days. Results The occurrence of NSF-like macroscopic skin lesions differed between the injection groups. Shorter injection intervals resulted in more severe skin reactions. In contrast, the injection interval did not influence the long-term presence and level of accumulation of Gd concentration in tissue. The single injection of Gd-DTPA-BMA was followed by a rapid and transient induction of signaling molecules in the serum (MCP1, MCP3, IL1, IP-10, Osteopontine SCF and Timp1) as well as in the skin (MCP1 and TGFb). Conclusion The presence of NSF-like fibrosing dermopathy in rats was found to be dependent on the injection interval and not on the amount of Gd in tissue. Our findings suggest the possibility of a more acute intrinsic reaction on administration of Gd-DTPA-BMA that triggers events leading to the development of skin lesions. The finding that single injections of Gd-DTPA-BMA were accompanied by a fast and transient induction of signaling molecules that are known to be involved in several fibrotic events provides additional support for this hypothesis. The study findings, however, do not support the theory that the long-term presence of Gd plays a relevant role in the development of NSF.

Based on an improvement in overall survival (OS) the multikinase inhibitor (MKI) sorafenib is currently approved for front-line therapy of unresectable hepatocellular carcinoma (HCC). Brivanib another MKI failed to improve OS compared to sorafenib (Johnson et al, 2013). In order to identify factors which discriminate sorafenib from other MKIs beyond their different kinase profiles, we initiated an exploratory study to compare the effects of sorafenib with brivanib on the liver of healthy rats in vivo. Since the liver exhibits substantial metabolic and regenerative abilities, measureable molecular effects of MKIs were thought to become apparent using transcript profiling. Six male Wistar rats each were treated once daily with either sorafenib or brivanib at doses corresponding to respective clinically relevant doses, or corresponding vehicles. Liver samples were collected three hours after two and 15 treatments for transcript profiling on Rat ClariomD microarrays. Data were analyzed and statistically evaluated using Genedata ProfilerTM software. Genes with a minimum 1.5-fold change vs control were considered relevant. In addition, drug pharmacokinetics was analyzed from plasma after nine treatments. Clinical pathology data indicated minor effects on liver function for both compounds, consistent with previous observations for sorafenib. Mild histological changes in liver, pancreas and spleen were observed for brivanib. Sorafenib exposure was approximately 5-times lower than expected from former rat studies. Changes in gene expression were detectable in livers from treated vs. control animals supporting our hypothesis. The number of deregulated genes and the extent of deregulation were less pronounced for sorafenib probably attributed to its lower exposure. Transcript profile changes by both compounds were measurable early after two treatments and became stronger after 15 treatments. Both drugs induced changes in the expression of genes involved in vascular functions, consistent with their inhibition of VEGFR kinase, interferon / interleukin signaling, and of genes affecting metabolic pathways. Sorafenib, despite its lower exposure, selectively altered expression of genes involved in cell cycle progression and genes potentially promoting differentiation, such as vestigial-like family member 4. Vgll4 has been described as tumor suppressor by negatively regulating the formation of the YAP-TEAD transcriptional complex. In summary, the use of transcript profiling of liver tissue from healthy rats enabled us to identify genes whose expression is differentially regulated by sorafenib and brivanib and which have reasonable functional relevance. Further studies including more sophisticated models will be performed to confirm and expand these results. Citation Format: Heidrun Ellinger-Ziegelbauer, Lukas Fiebig, Ludwig Schladt, Karl Ziegelbauer, Anette Sommer, Dieter Zopf. Differentiation of multi-kinase inhibitors by in vivo transcript profiling: Potential implications for HCC therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3935.

This study was designed to assess the value of a set of potential markers for improved detection of liver injury in preclinical toxicity studies. Male Wistar rats were treated with drug candidates (BAY16, EMD335823, BI-3) that previously failed during development, in part due to hepatotoxicity, at two dose levels for 1, 3 and 14 days. Concentrations of lipocalin-2/NGAL and clusterin, which are frequently overexpressed and released from damaged tissues, and thiostatin, recently identified within PredTox as being elevated in urine in response to liver injury, were determined in rat urine and serum by ELISA. This was supplemented by confirmatory qRT-PCR and immunohistochemical analyses in the target organ. Serum paraoxonase-1 activity (PON1), which has been suggested as a marker of hepatotoxicity, was determined using a fluorometric assay. Clusterin and PON1 were not consistently altered in response to liver injury. In contrast, thiostatin and NGAL were increased in serum and urine of treated animals in a time- and dose-dependent manner. These changes correlated well with mRNA expression in the target organ and generally reflected the onset and degree of drug-induced liver injury. Receiver-operating characteristics (ROC) analyses supported serum thiostatin, but not NGAL, as a better indicator of drug-induced hepatobiliary injury than conventional clinical chemistry parameters, i.e. ALP, ALT and AST. Although thiostatin, an acute phase protein expressed in a range of tissues, may not be specific for liver injury, our results indicate that thiostatin may serve as a sensitive, minimally-invasive diagnostic marker of inflammation and tissue damage in preclinical safety assessment.

Manufactured multi-walled carbon nanotubes (MWCNT) have attracted a great deal of attention due to their unique structural, chemical, and physical characteristics. This study utilized a 1x 6h inhalation exposure protocol followed by a 3 months post-exposure period. Wistar rats were nose-only exposed to 11 and 241 mg/m(3) MWCNT (Baytubes) of respirable, solid aerosol. MWCNT depleted of residual metals (depletion from 0.53% to 0.12% Co) were compared at 11 mg/m(3). Rats similarly exposed to air and alpha-quartz (248 mg/m(3)) served as negative and positive controls, respectively. Pulmonary response was characterized by bronchoalveolar lavage (BAL), lung histopathology, organ burden determinations, and gene expression analyses of lung homogenates with emphasis on extracellular matrix components. This acute inhalation exposure protocol was suitable to characterize and distinguish acute deposition-related effects from the long-term sequelae of retained MWCNT. Subtle differences in acute pulmonary toxic potency due to differences in metal contaminations could be revealed by this protocol. Consistent with the long retention halftime of poorly soluble particles, even short-term inhalation studies may require post-exposure periods of at least 3 months to reveal MWCNT-specific dispositional and toxicological characteristics relative to alpha-quartz. Distinct differences in the time course of pulmonary inflammation of MWCNT and alpha-quartz could be demonstrated. Transcriptomics proved to be a useful tool to analyze the etiopathology of collagen detected by BAL and histopathology. In summary, the pulmonary inflammogenicity following exposure to MWCNT was concentration-dependent with evidence of regression over time. Conversely, alpha-quartz resulted in progressive changes over time. The time course of pulmonary inflammation associated with retained MWCNT was independent on the concentration of residual cobalt. This supports the conclusion that the predominant response to inhaled MWCNT is principally related to the assemblage structure and not catalyst impurities (if in the range of < or = 0.5%).

The genotoxicity testing battery is highly sensitive for detection of chemical carcinogens. However, it features a low specificity and provides only limited mechanistic information required for risk assessment of positive findings. This is especially important in case of positive findings in the in vitro chromosome damage assays, because chromosome damage may be also induced secondarily to cell death. An increasing body of evidence indicates that toxicogenomic analysis of cellular stress responses provides an insight into mechanisms of action of genotoxicants. To evaluate the utility of such a toxicogenomic analysis we evaluated gene expression profiles of TK6 cells treated with four model genotoxic agents using a targeted high density real-time PCR approach in a multilaboratory project coordinated by the Health and Environmental Sciences Institute Committee on the Application of Genomics in Mechanism-based Risk Assessment. We show that this gene profiling technology produced reproducible data across laboratories allowing us to conclude that expression analysis of a relevant gene set is capable of distinguishing compounds that cause DNA adducts or double strand breaks from those that interfere with mitotic spindle function or that cause chromosome damage as a consequence of cytotoxicity. Furthermore, our data suggest that the gene expression profiles at early time points are most likely to provide information relevant to mechanisms of genotoxic damage and that larger gene expression arrays will likely provide richer information for differentiating molecular mechanisms of action of genotoxicants. Although more compounds need to be tested to identify a robust molecular signature, this study confirms the potential of toxicogenomic analysis for investigation of genotoxic mechanisms.

The carcinogenic potential of chemicals is currently evaluated with rodent life-time bioassays, which are time consuming, and expensive with respect to cost, number of animals and amount of compound required. Since the results of these 2-year bioassays are not known until quite late during development of new chemical entities, and since the short-term test battery to test for genotoxicity, a characteristic of genotoxic carcinogens, is hampered by low specificity, the identification of early biomarkers for carcinogenicity would be a big step forward. Using gene expression profiles from the livers of rats treated up to 14 days with genotoxic and non-genotoxic carcinogens we previously identified characteristic gene expression profiles for these two groups of carcinogens. We have now added expression profiles from further hepatocarcinogens and from non-carcinogens the latter serving as control profiles. We used these profiles to extract biomarkers discriminating genotoxic from non-genotoxic carcinogens and to calculate classifiers based on the support vector machine (SVM) algorithm. These classifiers then predicted a set of independent validation compound profiles with up to 88% accuracy, depending on the marker gene set. We would like to present this study as proof of the concept that a classification of carcinogens based on short-term studies may be feasible.

Application of recently developed gene expression techniques using microarrays in toxicological studies (toxicogenomics) facilitate the interpretation of a toxic compound's mode of action and may also allow the prediction of selected toxic effects based on gene expression changes. In order to test this hypothesis, we investigated whether carcinogens at doses known to induce liver tumors in the 2-year rat bioassay deregulate characteristic sets of genes in a short term in vivo study and whether these deregulated genes represent defined biological pathways. Male Wistar rats were dosed with the four nongenotoxic hepatocarcinogens methapyrilene (MPy, 60 mg/kg/day), diethylstilbestrol (DES, 10 mg/kg/day), Wy-14643 (Wy, 60 mg/kg/day), and piperonylbutoxide (PBO, 1200 mg/kg/day). After 1, 3, 7, and 14 days, the livers were taken for histopathological evaluation and for analysis of the gene expression profiles on Affymetrix RG_U34A arrays. The expression profile of the four nongenotoxic carcinogens were compared to the profiles of the four genotoxic carcinogens 2-nitrofluorene (2-NF), dimethylnitrosamine (DMN), 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), and aflatoxin B1 (AB1) from a similar study reported previously. By using statistical and clustering tools characteristically deregulated genes were extracted and functionally classified. Distinct cellular pathways were affected by the nongenotoxic carcinogens compared to the genotoxic carcinogens which at least partly correlated with the two-stage model of carcinogenesis. Characteristic to genotoxic carcinogens were a DNA damage response and the activation of proliferative and survival signaling. Nongenotoxic carcinogens showed responses to oxidative DNA or protein damage, as well as cell cycle progression and signs of regeneration. Many of the gene alterations found with the nongenotoxic carcinogens imply compound-specific mechanisms. Although neither a single gene nor a single pathway will be sufficient to discriminate the two classes of carcinogens, it became evident that combinations of pathway-associated gene expression profiles may be used to predict a genotoxic or nongenotoxic carcinogenic potential of a compound in short-term studies.

When applied in toxicological studies, the recently developed gene expression profiling techniques using microarrays, which brought forth the new field of toxicogenomics, facilitate the interpretation of a toxic compound's mechanism of action. In this study, we investigated whether genotoxic carcinogens at doses known to induce liver tumors in the 2-year rat bioassay deregulate a common set of genes in a short-term in vivo study and, if so, whether these deregulated genes represent defined biological pathways. Rats were dosed with the four genotoxic hepatocarcinogens dimethylnitrosamine (4 mg/kg/day), 2-nitrofluorene (44 mglkg/ day), aflatoxin B1 (0.24 mg/kg/day), and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK, 20 mg/kg/day). After treatment for up to 14 days, the expression profiles of the livers were analyzed on Affymetrix RG_U34A microarrays. Among the significantly upregulated genes were a set of target genes of the tumor suppressor protein p53, indicating a DNA damage response. Such a response was expected and, therefore, confirmed the validity of our approach. In addition, the gene expression changes suggest a specific detoxification response, the activation of proliferative and survival signaling pathways, and some cell structural changes. These responses were strong throughout the 14 day time course for 2-nitrofluorene and aflatoxin B1; in the case of dimethylnitrosamine and NNK, the effects were weakly detectable at day 1 and then increased with time. For dimethylnitrosamine and aflatoxin B1, which caused observable inflammation in vivo, we found a corresponding upregulation of inflammatory genes at the same time points. Thus, by the toxicogenomic analysis of short-term in vivo studies, we identified genes and pathways commonly deregulated by genotoxic carcinogens, which may be indicative for the early events in tumorigenesis and, thus, predictive of later tumor development.