Gene expression of mesothelioma in vinylidene chloride-exposed F344/N rats reveal immune dysfunction, tissue damage, and inflammation pathways.

A majority (∼80%) of human malignant mesotheliomas are asbestos-related. However, non-asbestos risk factors (radiation, chemicals, and genetic factors) account for up to 30% of cases. A recent 2-year National Toxicology Program carcinogenicity bioassay showed that male F344/N rats exposed to the industrial toxicant vinylidene chloride (VDC) resulted in a marked increase in malignant mesothelioma. Global gene expression profiles of these tumors were compared to spontaneous mesotheliomas and the F344/N rat mesothelial cell line (Fred-PE) in order to characterize the molecular features and chemical-specific profiles of mesothelioma in VDC-exposed rats. As expected, mesotheliomas from control and VDC-exposed rats shared pathways associated with tumorigenesis, including cellular and tissue development, organismal injury, embryonic development, inflammatory response, cell cycle regulation, and cellular growth and proliferation, while mesotheliomas from VDC-exposed rats alone showed overrepresentation of pathways associated with pro-inflammatory pathways and immune dysfunction such as the nuclear factor kappa-light-chain-enhancer of activated B cells signaling pathway, interleukin (IL)-8 and IL-12 signaling, interleukin responses, Fc receptor signaling, and natural killer and dendritic cells signaling, as well as overrepresentation of DNA damage and repair. These data suggest that a chronic, pro-inflammatory environment associated with VDC exposure may exacerbate disturbances in oncogene, growth factor, and cell cycle regulation, resulting in an increased incidence of mesothelioma.

Spontaneous mesotheliomas in F344/N rats are characterized by dysregulation of cellular growth and immune function pathways.

Aged male Fischer 344/N rats are prone to developing spontaneous peritoneal mesotheliomas that arise predominantly from the tunica vaginalis of the testes. A definitive cause for the predominance of this neoplasm in F344/N rats is unknown. Investigation of the molecular alterations that occur in spontaneous rat mesotheliomas may provide insight into their pathogenesis as well enable a better understanding regarding the mechanisms underlying chemically induced mesothelioma in rodents. Mesothelial cell function represents a complex interplay of pathways related to host defense mechanisms and maintenance of cellular homeostasis. Global gene expression profiles of spontaneous mesotheliomas from vehicle control male F344/N rats from 2-year National Toxicology Program carcinogenicity bioassays were analyzed to determine the molecular features of these tumors and elucidate tumor-specific gene expression profiles. The resulting gene expression pattern showed that spontaneous mesotheliomas are associated with upregulation of various growth factors, oncogenes, cytokines, pattern recognition response receptors, and pathogen-associated molecular patterns receptors, and the production of reactive oxygen and nitrogen species, as well as downregulation of apoptosis pathways. Alterations in these pathways in turn trigger molecular responses that stimulate cell proliferation and promote tumor survival and progression.

Inhibition of the thyroid hormone pathway in Xenopus laevis by 2-mercaptobenzothiazole.

Determining the effects of chemicals on the thyroid system is an important aspect of evaluating chemical safety from an endocrine disrupter perspective. Since there are numerous chemicals to test and limited resources, prioritizing chemicals for subsequent in vivo testing is critical. 2-Mercaptobenzothiazole (MBT), a high production volume chemical, was tested and shown to inhibit thyroid peroxidase (TPO) enzyme activity in vitro, a key enzyme necessary for the synthesis of thyroid hormone. To determine the thyroid disrupting activity of MBT in vivo, Xenopus laevis larvae were exposed using 7- and 21-day protocols. The 7-day protocol used 18-357 µg/L MBT concentrations and evaluated: metamorphic development, thyroid histology, circulating T4, circulating thyroid stimulating hormone, thyroidal sodium-iodide symporter gene expression, and thyroidal T4, T3, and related iodo-amino acids. The 21-day protocol used 23-435 µg/L MBT concentrations and evaluated metamorphic development and thyroid histology. Both protocols demonstrated that MBT is a thyroid disrupting chemical at the lowest concentrations tested. These studies complement the in vitro study used to identify MBT as a high priority for in vivo testing, supporting the utility/predictive potential of a tiered approach to testing chemicals for TPO activity inhibition. The 7-day study, with more comprehensive, sensitive, and diagnostic endpoints, provides information at intermediate biological levels that enables linking various endpoints in a robust and integrated pathway for thyroid hormone disruption associated with TPO inhibition.

Trenbolone causes mortality and altered sexual differentiation in Xenopus tropicalis during larval development.

Trenbolone is an androgen agonist used in cattle production and has been measured in aquatic systems associated with concentrated animal-feeding operations. In this study, the authors characterized the effects of aqueous exposure to 17ß-trenbolone during larval Xenopus tropicalis development. Trenbolone exposure resulted in increased mortality of post-Nieuwkoop-Faber stage 58 tadpoles at concentrations ≥100 ng/L. Morphological observations and the timing of this mortality are consistent with hypertrophy of the larynx. Development of nuptial pads, a male secondary sex characteristic, was induced in tadpoles of both sexes at 100 ng/L. Effects on time to complete metamorphosis or body sizes were not observed; however, grow-outs placed in clean media for six weeks were significantly smaller in body size at 78 ng/L. Effects on sex ratios were equivocal, with the first experiment showing a significant shift in sex ratio toward males at 78 ng/L. In the second experiment, no significant effects were observed up to 100 ng/L, although overall sex ratios were similar. Histological assessment of gonads at metamorphosis showed half with normal male phenotypes and half that possessed a mixed-sex phenotype at 100 ng/L. Hypertrophy of the Wolffian ducts was also observed at this concentration. These results indicate that larval 17ß-trenbolone exposure results in effects down to 78 ng/L, illustrating potential effects from exposure to androgenic compounds in anurans.

Proceedings of the 2010 National Toxicology Program Satellite Symposium.

The 2010 annual National Toxicology Program (NTP) Satellite Symposium, entitled "Pathology Potpourri," was held in Chicago, Illinois, in advance of the scientific symposium sponsored jointly by the Society of Toxicologic Pathology (STP) and the International Federation of Societies of Toxicologic Pathologists (IFSTP). The goal of the annual NTP Symposium is to present current diagnostic pathology or nomenclature issues to the toxicologic pathology community. This article presents summaries of the speakers' presentations, including diagnostic or nomenclature issues that were presented, along with select images that were used for voting or discussion. Some topics covered during the symposium included a comparison of rat and mouse hepatocholangiocarcinoma, a comparison of cholangiofibrosis and cholangiocarcinoma in rats, a mixed pancreatic neoplasm with acinar and islet cell components, an unusual preputial gland tumor, renal hyaline glomerulopathy in rats and mice, eosinophilic substance in the nasal septum of mice, INHAND nomenclature for proliferative and nonproliferative lesions of the CNS/PNS, retinal gliosis in a rat, fibroadnexal hamartoma in rats, intramural plaque in a mouse, a treatment-related chloracne-like lesion in mice, and an overview of mouse ovarian tumors.

Fourteen-week toxicity study of green tea extract in rats and mice.

The toxicity of green tea extract (GTE) was evaluated in 14-week gavage studies in male and female F344/NTac rats and B6C3F1 mice at doses up to 1,000 mg/kg. In the rats, no treatment-related mortality was noted. In the mice, treatment-related mortality occurred in male and female mice in the 1,000 mg/kg dose groups. The cause of early deaths was likely related to liver necrosis. Treatment-related histopathological changes were seen in both species in the liver, nose, mesenteric lymph nodes, and thymus. In addition, in mice, changes were seen in the Peyer's patches, spleen, and mandibular lymph nodes. The no adverse effect level (NOAEL) for the liver in both species was 500 mg/kg. In the nose of rats, the NOAEL in males was 62.5 mg/kg, and in females no NOAEL was found. No NOAEL was found in the nose of female or male mice. The changes in the liver and nose were considered primary toxic effects of GTE, while the changes in other organs were considered to be secondary effects. The nose and liver are organs with high metabolic enzyme activity. The increased susceptibility of the nose and liver suggests a role for GTE metabolites in toxicity induction.

Gene expression response in target organ and whole blood varies as a function of target organ injury phenotype.

This report details the standardized experimental design and the different data streams that were collected (histopathology, clinical chemistry, hematology and gene expression from the target tissue (liver) and a bio-available tissue (blood)) after treatment with eight known hepatotoxicants (at multiple time points and doses with multiple biological replicates). The results of the study demonstrate the classification of histopathological differences, likely reflecting differences in mechanisms of cell-specific toxicity, using either liver tissue or blood transcriptomic data.

Hepatic transcript levels for genes coding for enzymes associated with xenobiotic metabolism are altered with age.

Metabolism studies are crucial for data interpretation from rodent toxicity and carcinogenicity studies. Metabolism studies are usually conducted in 6 to 8 week old rodents. Long-term studies often continue beyond 100 weeks of age. The potential for age-related changes in transcript levels of genes encoding for enzymes associated with metabolism was evaluated in the liver of male F344/N rats at 32, 58, and 84 weeks of age. Differential expression was found between the young and old rats for genes whose products are involved in both phase I and phase II metabolic pathways. Thirteen cytochrome P450 genes from CYP families 1-3 showed alterations in expression in the older rats. A marked age-related decrease in expression was found for 4 members of the Cyp3a family that are critical for drug metabolism in the rat. Immunohistochemical results confirmed a significant decrease in Cyp3a2 and Cyp2c11 protein levels with age. This indicates that the metabolic capacity of male rats changes throughout a long-term study. Conducting multiple hepatic microarray analyses during the conduct of a long-term study can provide a global view of potential metabolic changes that might occur. Alterations that are considered crucial to the interpretation of long-term study results could then be confirmed by subsequent metabolic studies.

Application of visualization tools to the analysis of histopathological data enhances biological insight and interpretation.

Gene expression profiling, metabolomic screens, and other high-dimensional methods have become an integral part of many biological investigations. To facilitate interpretation of these data, it is important to have detailed phenotypic data--including histopathology--to which these data can be associated, or anchored. However, as the amount of phenotypic data increases, associations within and across these data can be difficult to visualize and interpret. We have developed an approach for categorizing and clustering biologically related histopathological diagnoses to facilitate their visualization, thereby increasing the possibility of identifying associations and facilitating the comparison with other data streams. In this study, we utilize histopathological data generated as part of a standardized toxicogenomics compendium study to generate composite histopathological scores and to develop visualizations that facilitate biological insight. The validity of this approach is illustrated by the identification of transcripts that correlate with the pathology diagnoses that comprise the categories of "response to hepatocellular injury" and "repair." This approach is broadly applicable to studies in which histopathology is used to phenotypically anchor other data, and results in visualizations that facilitate biological interpretation and the identification of associations and relationships within the data.

Transcriptional profiling of the left and median liver lobes of male f344/n rats following exposure to acetaminophen.

The liver is a common organ for transcriptional profiling because of its role in xenobiotic metabolism and because hepatotoxicity is a common response to chemical exposure. To explore the impact that sampling different lobes may have on transcriptional profiling experiments we have examined and compared gene expression profiles of the left and median lobes of livers from male F344 rats exposed to toxic and nontoxic doses of acetaminophen. Transcript profiling using micorarrays revealed clear differences in the response of the left and median liver lobes of F344 rats to acetaminophen exposure both at low doses as well as doses that caused hepatotoxicity. Differences were found in the total number of differentially expressed genes in the left and median lobes, the number and identity of genes that were differentially expressed uniquely only in the left or median lobe, and in the patterns of gene expression. While it is not possible to generalize these results to compounds other than acetaminophen or other strains of rat, these results highlight the potential impact of sampling differences on the interpretation of gene expression profiles in the liver.

Hepatic gene expression changes throughout the day in the Fischer rat: implications for toxicogenomic experiments.

There is increasing use of transcriptional profiling in hepatotoxicity studies in the rat. Understanding hepatic gene expression changes over time is critical, since tissue collection may occur throughout the day. Furthermore, when comparing results from different data sets, times of dosing and tissue collection may vary. Circadian effects on the mouse hepatic transcriptome have been well documented. However, limited reports exist for the rat. In one study approximately 7% of the hepatic genes showed a diurnal expression pattern in a comparison of rat liver samples collected during the day versus livers collected at night. The results of a second study comparing rat liver samples collected at multiple time points over a circadian day suggest only minimal variation of the hepatic transcriptome. We studied temporal hepatic gene expression in 48 untreated F344/N rats using both approaches employed in these previous studies. Statistical analysis of microarray (SAM) identified differential expression in day/night comparisons, but was less sensitive for liver samples collected at multiple times of day. However, a Fourier analysis identified numerous periodically expressed genes in these samples including period genes, clock genes, clock-controlled genes, and genes involved in metabolic pathways. Furthermore, rhythms in gene expression were identified for several circadian genes not previously reported in the rat liver. Transcript levels for twenty genes involved in circadian and metabolic pathways were confirmed using quantitative RT-PCR. The results of this study demonstrate a prominent circadian rhythm in gene expression in the rat that is a critical factor in planning toxicogenomic experiments.

The toxicity of SCH 351591, a novel phosphodiesterase-4 inhibitor, in Cynomolgus monkeys.

SCH351591, a novel phosphodiesterase-4 inhibitor under investigation as a potential therapeutic for asthma and chronic obstructive pulmonary disease (COPD), was evaluated in a 3-month rising-dose study in Cynomolgus monkeys. Four groups, containing four monkeys/sex, received vehicle control or rising doses up to 12, 24, or 48 mg/kg of SCH351591 daily. Although initial exposure produced clinical signs of emesis, reduced food intake, and reduced body weight, tachyphylaxis to the emesis allowed dose escalation up to 48 mg/kg/day. Two monkeys died and 3 were sacrificed in moribund condition over the course of the study. Early mortality, involving monkeys dosed with 12 or 24 mg/kg, was attributed to sepsis (2 monkeys) or colon inflammation (3 monkeys). Leukocyte function assays on low- and mid-dose group survivors revealed an inhibition of T lymphocyte proliferation for 12 mg/kg group males and 24 mg/kg group monkeys of both sexes. Necropsy findings, unassociated with early mortality, included reduced size and weight of the thymus, depletion of body fat, red discoloration of the gastric mucosa, and perivascular hemorrhage of the stomach and heart. Stomach and heart gross findings were present in the high-dose group only. Histopathologic lesions, in addition to those attributed to concurrent bacterial infection, included thymic atrophy, serous atrophy of fat, myocardial degeneration and acute to chronic inflammation of small to medium-sized arteries in various organs and tissues including the heart, kidneys, stomach, salivary glands, pancreas, esophagus, gallbladder, and mesentery. The findings of this study demonstrate the potential of a PDE4 inhibitor to alter immunologic response as well as to produce arteriopathy in nonhuman primates.

Gene expression profiling of rat livers reveals indicators of potential adverse effects.

This study tested the hypothesis that gene expression profiling can reveal indicators of subtle injury to the liver induced by a low dose of a substance that does not cause overt toxicity as defined by conventional criteria of toxicology (e.g., abnormal clinical chemistry and histopathology). For the purpose of this study we defined this low dose as subtoxic, i.e., a dose that elicits effects which are below the detection of conventional toxicological parameters. Acetaminophen (APAP) was selected as a model hepatotoxicant because (1) considerable information exists concerning the mechanism of APAP hepatotoxicity that can occur following high doses, (2) intoxication with APAP is the leading cause of emergency room visits involving acute liver failure within the United States, and (3) conventional clinical markers have poor predictive value. Rats treated with a single dose of 0, 50, 150, or 1500 mg/kg APAP were examined at 6, 24, or 48 h after exposure for conventional toxicological parameters and for gene expression alterations. Patterns of gene expression were found which indicated cellular energy loss as a consequence of APAP toxicity. Elements of these patterns were apparent even after exposure to subtoxic doses. With increasing dose, the magnitude of changes increased and additional members of the same biological pathways were differentially expressed. The energy loss suggested by gene expression changes was confirmed at the 1500 mg/kg dose exposure by measuring ATP levels. Only by ultrastructural examination could any indication of toxicity be identified after exposure to a subtoxic dose of APAP and that was occasional mitochondrial damage. In conclusion, this study provides evidence that supports the hypothesis that gene expression profiling may be a sensitive means of identifying indicators of potential adverse effects in the absence of the occurrence of overt toxicity.