Absence of Increased Susceptibility to Acetaminophen-Induced Liver Injury in a Diet-Induced NAFLD Mouse Model.

Nonalcoholic fatty liver disease (NAFLD) is a common chronic liver disease and its influence on drug-induced liver injury (DILI) is not fully understood. We investigated whether NAFLD can influence acetaminophen (APAP [N-acetyl-p-aminophenol])-induced hepatotoxicity in a diet-induced obese (DIO) mouse model of NAFLD. The male C57BL/6NTac DIO mice, fed a high-fat diet for more than 12 weeks, developed obesity, hyperinsulinemia, impaired glucose tolerance, and hepatomegaly with hepatic steatosis, similar to human NAFLD. In the acute toxicity study after a single dose of APAP (150 mg/kg), compared with control lean mice, the DIO mice had decreased serum transaminase levels and less severe hepatocellular injury. The DIO mice also had altered expression of genes related to APAP metabolism. Chronic APAP exposure for 26 weeks did not predispose the DIO mice with NAFLD to more severe hepatotoxicity compared with the lean mice. These results suggested that the C57BL/6NTac DIO mouse model appears to be more tolerant to APAP-induced hepatotoxicity than lean mice, potentially related to altered xenobiotic metabolizing capacity in the fatty liver. Further mechanistic studies with APAP and other drugs in NAFLD animal models are necessary to investigate the mechanism of altered susceptibility to intrinsic DILI in some human NAFLD patients.

Short-term tetrabromobisphenol A exposure promotes fibrosis of human uterine fibroid cells in a 3D culture system through TGF-beta signaling.

Tetrabromobisphenol A (TBBPA), a derivative of BPA, is a ubiquitous environmental contaminant with weak estrogenic properties. In women, uterine fibroids are highly prevalent estrogen-responsive tumors often with excessive accumulation of extracellular matrix (ECM) and may be the target of environmental estrogens. We have found that BPA has profibrotic effects in vitro, in addition to previous reports of the in vivo fibrotic effects of BPA in mouse uterus. However, the role of TBBPA in fibrosis is unclear. To investigate the effects of TBBPA on uterine fibrosis, we developed a 3D human uterine leiomyoma (ht-UtLM) spheroid culture model. Cell proliferation was evaluated in 3D ht-UtLM spheroids following TBBPA (10-6 -200 µM) administration at 48 h. Fibrosis was assessed using a Masson's Trichrome stain and light microscopy at 7 days of TBBPA (10-3  µM) treatment. Differential expression of ECM and fibrosis genes were determined using RT² Profiler™ PCR arrays. Network and pathway analyses were conducted using Ingenuity Pathway Analysis. The activation of pathway proteins was analyzed by a transforming growth factor-beta (TGFB) protein array. We found that TBBPA increased cell proliferation and promoted fibrosis in 3D ht-UtLM spheroids with increased deposition of collagens. TBBPA upregulated the expression of profibrotic genes and corresponding proteins associated with the TGFB pathway. TBBPA activated TGFB signaling through phosphorylation of TGFBR1 and downstream effectors-small mothers against decapentaplegic -2 and -3 proteins (SMAD2 and SMAD3). The 3D ht-UtLM spheroid model is an effective system for studying environmental agents on human uterine fibrosis. TBBPA can promote fibrosis in uterine fibroid through TGFB/SMAD signaling.

Preparation of Three-dimensional (3-D) Human Liver (HepaRG) Cultures for Histochemical and Immunohistochemical Staining and Light Microscopic Evaluation.

The use of three-dimensional (3-D) in vitro culture systems (spheroids, organoids) in biomolecular and drug discovery research has become increasingly popular. The popularity is due, in part, to a diminished reliance on animal bioassays and a desire to develop physiologically relevant cell culture systems that simulate the in vivo tissue microenvironment. Most evaluations of 3-D cultures are by confocal microscopy and high-content imaging; however, these technologies do not allow for detailed cellular morphologic assessments or permit basic hematoxylin and eosin histologic evaluations. There are few studies that have reported detailed processes for preparing 3-D cultures for paraffin embedding and subsequent use for histochemical or immunohistochemical staining. In an attempt to do so, we have developed a protocol to paraffin-embed human liver spheroids that can be sectioned with a microtome and mounted onto glass slides for routine histochemical and immunohistochemical staining and light microscopic evaluations.

Immunohistochemistry in Investigative and Toxicologic Pathology.

Immunohistochemistry (IHC) is a valuable tool in pathology. This review provides a brief description of the technical aspects of IHC and a detailed discussion on the variables that affect the results, interpretation, and reproducibility of IHC results. Lists of antibodies that have and have not worked in IHC on various mouse and rat tissues in our laboratory are provided as a guidance for selection of antibodies. An approach to IHC method optimization is presented. Finally, the critical information that should be included as a part of peer-reviewed manuscript is also discussed.

Renal Cell Carcinomas in Vinylidene Chloride-exposed Male B6C3F1 Mice Are Characterized by Oxidative Stress and TP53 Pathway Dysregulation.

Vinylidene chloride (VDC) has been widely used in the production of plastics and flame retardants. Exposure of B6C3F1 mice to VDC in the 2-year National Toxicology Program carcinogenicity bioassay resulted in a dose-dependent increases in renal cell hyperplasia, renal cell adenoma, and renal cell carcinomas (RCCs). Among those differentially expressed genes from controls and RCC of VDC-exposed mice, there was an overrepresentation of genes from pathways associated with chronic xenobiotic and oxidative stress as well as c-Myc overexpression and dysregulation of TP53 cell cycle checkpoint and DNA damage repair pathways in RCC. Trend analysis comparing RCC, VDC-exposed kidney, and chamber control kidney showed a conservation of pathway dysregulation in terms of overrepresentation of xenobiotic and oxidative stress, and DNA damage and cell cycle checkpoint pathways in both VDC-exposed kidney and RCC, suggesting that these mechanisms play a role in the pathogenesis of RCC in VDC-exposed mice.

Histopathological and Immunohistochemical Characterization of Methyl Eugenol-induced Nonneoplastic and Neoplastic Neuroendocrine Cell Lesions in Glandular Stomach of Rats.

Methyl eugenol induces neuroendocrine (NE) cell hyperplasia and tumors in F344/N rat stomach. Detailed histopathological and immunohistochemical (IHC) characterization of these tumors has not been previously reported. The objective of this study was to fill that data gap. Archived slides and paraffin blocks were retrieved from the National Toxicology Program Archives. NE hyperplasias and tumors were stained with chromogranin A, synaptophysin, amylase, gastrin, H(+)/K(+) adenosine triphosphatase (ATPase), pepsinogen, somatostatin, and cytokeratin 18 (CK18) antibodies. Many of the rats had gastric mucosal atrophy, due to loss of chief and parietal cells. The hyperplasias and tumors were confined to fundic stomach, and females were more affected than the males. Hyperplasia of NE cells was not observed in the pyloric region. Approximately one-third of the females with malignant NE tumors had areas of pancreatic acinar differentiation. The rate of metastasis was 21%, with liver being the most common site of metastasis. Immunohistochemically, the hyperplasias and tumors stained consistently with chromogranin A and synaptophysin. Neoplastic cells were also positive for amylase and CK18 and negative for gastrin, somatostatin, H(+)/K(+) ATPase, and pepsinogen. Metastatic neoplasms histologically similar to the primary neoplasm stained positively for chromogranin A and synaptophysin. Based on the histopathological and IHC features, the neoplasms appear to arise from enterochromaffin-like cells.

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.

Characterization of four new mouse cytochrome P450 enzymes of the CYP2J subfamily.

The cytochrome P450 superfamily encompasses a diverse group of enzymes that catalyze the oxidation of various substrates. The mouse CYP2J subfamily includes members that have wide tissue distribution and are active in the metabolism of arachidonic acid (AA), linoleic acid (LA), and other lipids and xenobiotics. The mouse Cyp2j locus contains seven genes and three pseudogenes located in a contiguous 0.62 megabase cluster on chromosome 4. We describe four new mouse CYP2J isoforms (designated CYP2J8, CYP2J11, CYP2J12, and CYP2J13). The four cDNAs contain open reading frames that encode polypeptides with 62-84% identity with the three previously identified mouse CYP2Js. All four new CYP2J proteins were expressed in Sf21 insect cells. Each recombinant protein metabolized AA and LA to epoxides and hydroxy derivatives. Specific antibodies, mRNA probes, and polymerase chain reaction primer sets were developed for each mouse CYP2J to examine their tissue distribution. CYP2J8 transcripts were found in the kidney, liver, and brain, and protein expression was confirmed in the kidney and brain (neuropil). CYP2J11 transcripts were most abundant in the kidney and heart, with protein detected primarily in the kidney (proximal convoluted tubules), liver, and heart (cardiomyocytes). CYP2J12 transcripts were prominently present in the brain, and CYP2J13 transcripts were detected in multiple tissues, with the highest expression in the kidney. CYP2J12 and CYP2J13 protein expression could not be determined because the antibodies developed were not immunospecific. We conclude that the four new CYP2J isoforms might be involved in the metabolism of AA and LA to bioactive lipids in mouse hepatic and extrahepatic tissues.

Genetic alterations in K-ras and p53 cancer genes in lung neoplasms from B6C3F1 mice exposed to cumene.

The incidences of alveolar/bronchiolar adenomas and carcinomas in cumene-treated B6C3F1 mice were significantly greater than those of the control animals. We evaluated these lung neoplasms for point mutations in the K-ras and p53 genes that are often mutated in humans. K-ras and p53 mutations were detected by cycle sequencing of PCR-amplified DNA isolated from paraffin-embedded neoplasms. K-ras mutations were detected in 87% of cumene-induced lung neoplasms, and the predominant mutations were exon 1 codon 12 G to T transversions and exon 2 codon 61 A to G transitions. P53 protein expression was detected by immunohistochemistry in 56% of cumene-induced neoplasms, and mutations were detected in 52% of neoplasms. The predominant mutations were exon 5, codon 155 G to A transitions, and codon 133 C to T transitions. No p53 mutations and one of seven (14%) K-ras mutations were detected in spontaneous neoplasms. Cumene-induced lung carcinomas showed loss of heterozygosity (LOH) on chromosome 4 near the p16 gene (13%) and on chromosome 6 near the K-ras gene (12%). No LOH was observed in spontaneous carcinomas or normal lung tissues examined. The pattern of mutations identified in the lung tumors suggests that DNA damage and genomic instability may be contributing factors to the mutation profile and development of lung cancer in mice exposed to cumene.

Gene expression studies demonstrate that the K-ras/Erk MAP kinase signal transduction pathway and other novel pathways contribute to the pathogenesis of cumene-induced lung tumors.

National Toxicology Program (NTP) inhalation studies demonstrated that cumene significantly increased the incidence of alveolar/bronchiolar adenomas and carcinomas in B6C3F1 mice. Cumene or isopropylbenzene is a component of crude oil used primarily in the production of phenol and acetone. The authors performed global gene expression analysis to distinguish patterns of gene regulation between cumene-induced tumors and normal lung tissue and to look for patterns based on the presence or absence of K-ras and p53 mutations in the tumors. Principal component analysis segregated the carcinomas into groups with and without K-ras mutations, but failed to separate the tumors based on p53 mutation status. Expression of genes associated with the Erk MAP kinase signaling pathway was significantly altered in carcinomas with K-ras mutations compared to tumors without K-ras mutations or normal lung. Gene expression analysis also suggested that cumene-induced carcinomas with K-ras mutations have greater malignant potential than those without mutations. In addition, significance analysis of function and expression (SAFE) demonstrated expression changes of genes regulated by histone modification in carcinomas with K-ras mutations. The gene expression analysis suggested the formation of alveolar/bronchiolar carcinomas in cumene-exposed mice typically involves mutation of K-ras, which results in increased Erk MAP kinase signaling and modification of histones.

Immunohistochemical analysis of expressions of hepatic cytochrome P450 in F344 rats following oral treatment with kava extract.

Kava (Piper methysticum), used for relaxation and pain relief, has been one of the leading dietary supplements and several reports linking hepatic functional disturbances and liver failure to kava have resulted in a ban on sales in Europe and Canada and the issuance of warnings by the US FDA. The National Toxicology Program conducted 14-week rat studies to characterize the toxicology of kava exposure in Fischer 344 rats [National Toxicity Program. 90 day gavage toxicity studies of KAVA KAVA EXTRACT in Fischer rats and B6C3F1 mice. Research Triangle Park, NC; 2005a; National Toxicity Program. Testing status of agents at NTP (KAVA KAVA EXTRACT M990058). Research Triangle Park, NC; 2005b. (http://ntp.niehs.nih.gov/index.cfm?objectid=071516E-C6E1-7AAA-C90C751E23D14C1B)]. Groups of 10 male and 10 female rats were administered kava extract by gavage at 0, 0.125, 0.25, 0.5, 1.0, and 2.0 g/kg/day. Increased gamma-glutamyl-transpeptidase (GGT) activities were observed in the 2.0 g/kg males and 1.0 and 2.0 g/kg females, as well as increased serum cholesterol levels in males and females at 0.5 g/kg and higher. Increases in incidence and severity of hepatocellular hypertrophy (HP) were noted in males at 1.0 g/kg and females at 0.5 g/kg and higher, as well as increased liver weights. Immunohistochemical analyses of the expression of cytochrome-P450 (CYP) enzymes in liver of the control and 1.0- and 2.0-g/kg-treated groups indicated decreased expression of CYP2D1 (human CYP2D6 homolog) in 2.0 g/kg females and increased expression of CYP1A2, 2B1, and 3A1 in 1.0 and 2.0 g/kg groups of both sexes. The no observed adverse effect levels were decided as 0.25 g/kg in both genders, based on neurotoxic effects, increases in GGT, cholesterol, liver weight, and HP and decreases in body weight. Kava-induced hepatic functional changes in the F344 rat might be relevant to human clinical cases of hepatotoxicity following exposure.

Chemical-induced atrial thrombosis in NTP rodent studies.

Cardiac thrombosis, one of the causes of sudden death throughout the world, plays a principal role in several cardiovascular diseases, such as myocardial infarction and stroke in humans. Data from studies of induction of chemical thrombosis in rodents help to identify substances in our environment that may contribute to cardiac thrombosis. Results for more than 500 chemicals tested in rodents in 2-year bioassays have been published as Technical Reports of the National Toxicology Program (NTP) http://ntp-server.niehs.nih.gov/index. We evaluated atrial thrombosis induced by these chemical exposures and compared it to similarly induced lesions reported in the literature. Spontaneous rates of cardiac thrombosis were determined for control Fischer 344 rats and B6C3F1 mice: 0% in rats and mice in 90-day studies and, in 2-year studies, 0.7% in both genders of mice, 4% in male rats, and 1% in female rats. Incidences of atrial thrombosis were increased in high-dosed groups involving 13 compounds (incidence rate: 20-100%): 2-butoxyethanol, C.I. Direct Blue 15, bis(2-chloroethoxy)methane, diazoaminobenzene, diethanolamine, 3,3'-dimethoxybenzidine dihydrochloride, hexachloroethane, isobutene, methyleugenol, oxazepam, C.I. Pigment Red 23, C.I. Acid Red 114, and 4,4'-thiobis(6-t-butyl-m-cresol). The main localization of spontaneously occurring and chemically induced thromboses occurred in the left atrium. The literature survey suggested that chemical-induced atrial thrombosis might be closely related to myocardial injury, endothelial injury, circulatory stasis, hypercoagulability, and impaired atrial mechanical activity, such as atrial fibrillation, which could cause stasis of blood within the left atrial appendage, contributing to left atrial thrombosis. Supplementary data referenced in this paper are not printed in this issue of Toxicologic Pathology. They are available as downloadable files at http://taylorandfrancis.metapress.com/openurl.asp?genre=journal&issn=0192-6233. To access them, click on the issue link for 33(5), then select this article. A download option appears at the bottom of this abstract. In order to access the full article online, you must either have an individual subscription or a member subscription accessed through www.toxpath.org.

Genetic alterations in brain tumors following 1,3-butadiene exposure in B6C3F1 mice.

The nervous system of the B6C3F1 mouse has rarely been a target for chemical carcinogenesis in the National Toxicology Program (NTP) bioassays. However, 6 malignant gliomas and 2 neuroblastomas were observed in B6C3F1 mice exposed to 625 ppm 1,3-butadiene (NTP technical reports 288 and 434). These mouse brain tumors were evaluated with regard to the profile of genetic alterations that are observed in human brain tumors. Alterations in the p53 tumor suppressor gene were common. Missense mutations were observed in 3/6 malignant gliomas and 2/2 neuroblastomas and were associated with loss of heterozygosity. Most of the mutations occurred in exons 5-8 of the p53 gene and were G-->A transitions, and did not involve CpG sites. Loss of heterozygosity at the Ink4a/Arf gene locus was observed in 5/5 malignant gliomas and 1/1 neuroblastoma, while the PTEN(phosphatase and tensin homologue) gene locus was unaffected by deletions. One of 2 neuroblastomas had a mutation in codon 61 of H-ras, while H-ras mutations were not observed in the malignant gliomas examined. Only 1 brain tumor has been reported from control mice of over 500 NTP studies. This malignant glioma showed no evidence of alterations in the p53 gene or K- and H-ras mutations. It is likely that the specific genetic alterations observed were induced or selected for by 1,3-butadiene treatment that contributed to the development of mouse brain tumors. The observed findings are similar in part to the genetic alterations reported in human brain tumors.