1. Hepatocholangiocarcinomas In B6C3F1 Mice
Rebecca Moore1, Gabrielle Willson1, David Malarkey2, Rodney Miller1, Grace Kissling3
1Experimental Pathology Laboratories, Inc., Research Triangle Park, NC, United States, 2Cellular and Molecular Pathology Branch, National Institute of Environmental Health Sciences, Research Triangle Park, NC, United States, 3Biostatistics Branch, National Institute of Environmental Health Sciences, Research Triangle Park, NC, United States
Hepatocholangiocarcinoma (HCCC) is a rare tumor with an overall incidence of 0.79% in male mice and 0.14% in female mice in the current National Toxicology Program (NTP) Historical Controls Database. HCCC is composed of a combination of neoplastic hepatocytes and neoplastic biliary epithelial cells. In addition to the hepatocellular and biliary epithelial components, there is often a poorly differentiated cellular component that has a sarcomatous to anaplastic and undifferentiated appearance. The variable histological morphology of the primary HCCC and the metastatic lesions can present a diagnostic challenge. The purpose of this retrospective study is to describe the morphological features, incidence, and behavior of HCCC in B6C3F1 mice. The NTP historical database from 1982 to 2006 was reviewed for a diagnosis of HCCC in B6C3F1 mice, and records and histologic slides of mice bearing HCCC were evaluated. A total of 164 HCCCs from 74 two-year carcinogenicity studies were found. They occurred more commonly in males (58%) than in females (42%). Metastases were present in 138 animals (84%) and often occurred in multiple sites. The sites most frequently observed to have metastatic lesions included lung (125/138, or 91%), mediastinum (104/138, or 75%), mesentery (80/138, or 58%), lymph nodes (71/138, or 51%), skeletal muscle (55/138, or 40%), kidney (47/138, or 34%), heart (46/138, or 33%), and pancreas (11/138, or 8%). HCCCs were not considered treatment-related in any study; however, in 24 of 74 studies (32%) there was a treatment-related increase in hepatocellular tumors in one or both sexes.
2. Predicting Rat Carcinogenicity Findings Using Chronic Study Results
Susan Turnquist, Daniel Morton
Pfizer Inc, Groton, CT, United States
The PhRMA PSLC Carcinogenicity Study Evaluation Working Group, a collaborative initiative involving 13 companies, was launched with the goal of improving speed and optimizing resource commitment to assess pharmaceutical carcinogenicity risk. Histopathology findings of foci of cellular alteration, hyperplasia and neoplasia in 6- and 12-month rat toxicity studies were compared to carcinogenicity findings in 2-year rat bioassays for 187 compounds. The presence of any positive proliferative findings in rat 6-month toxicity studies predicted positive findings in 2-year carcinogenicity studies for 59% of 59 compounds with positive carcinogenicity findings. Seventy-one percent of 83 compounds that had no proliferative microscopic lesions in any organs in 6-month studies had no treatment-related carcinogenicity findings. Twelve-month studies predicted positive and negative carcinogenicity findings slightly better than 6-month studies (63% sensitivity and 79% negative predictivity respectively), however compounds with 12-month study data were less common that those with 6-month data. Organ weight data from chronic toxicity studies and hormonal mechanisms of action will be evaluated to see if they improve power to predict carcinogenicity when combined with histopathology findings in chronic studies. The ability to predict negative carcinogenicity findings in 2-year rat studies with high confidence based on negative findings in 6-month chronic toxicity studies may justify conducting fewer 2-year rat bioassays.
3. Reported Medulloblastoma in the F344 Rat: National Toxicology Program (NTP) Experience
Deepa Rao1, James Morrison2, Hiroshi Satoh3, David Malarkey1
1National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina, United States, 2Charles River Laboratories, Pathology Associates, Durham, North Carolina, United States, 3Ina Research Inc., Tokyo, Japan
Medulloblastomas (MB) are malignant primitive embryonal tumors located within the cerebellum, and are the most common malignant tumor in the central nervous system (CNS) of children. Literature review reveals few cases of MBs in rats. The NTP historical control database between 2003 and 2008 indicates an incidence of 0.03%. The objectives of this retrospective study were to estimate the incidence, confirm the diagnoses, describe morphologic and immunohistochemical features, and report any clinical findings in rats with MBs from NTP studies conducted between 1971 and 2008. Twenty-four F344 rats from twenty-three different studies were identified with medulloblastomas, of which twenty were originally diagnosed as meningiomas. Eleven were found in control animals, thirteen in treated animals, and all were considered incidental. Fourteen cases were males. Clinical observations recorded in animals with medulloblastoma included abnormal posture, ataxia, hypoactivity, head tilt, and circling. All tumors were associated with or located within the cerebellum, and often extended into the fourth ventricle. Histologically, tumors were composed of closely packed neoplastic cells arranged as bundles and haphazardly interweaving streams with round, oval, conical or elongated nuclei, and scant cytoplasm. Mitotic figures were numerous. The intervening vascular stroma revealed prominent, multiple layered blood vessels. Typical Homer-Wright or Flexner-Wintersteiner rosette formations were not seen although occasional pseudo-rosettes were evident. Additionally, six tumors revealed nodular/desmoplastic features as reported in human medulloblastomas. This is the first comprehensive study of rat MBs. Results demonstrate that MBs are rare, usually incidental, can be associated with clinical signs, and are often misdiagnosed as meningiomas.
4. A Comparison of the Effects of the Rodent Liver Carcinogens on Rat Liver under ad libitum Feeding and Food Restriction
Takayuki Tsuchiya1, Chie Okumura1, Hiroki Sugiura1, Forest Thomas2, Minoru Sasaki1, Gordon Wollenberg2, Prahalada Srinvasa2
1Banyu Pharmaceutical Co., Ltd., Tsukuba, Japan, 2Merck Research Laboratories, West Point, United States
Food restriction (FR) in chronic toxicity studies or carcinogenicity studies in rats has advantages in terms of delayed onset of background histologic lesions and lower mortality compare to those in rats fed ad libitum. While FR has been known to alter cell proliferation in variety of organs including liver, a comparison of response to rodent liver carcinogens between ad lib feeding (ad lib) and FR has not been well addressed. The objective of this study is to confirm if the effects of the rodent liver carcinogens on rat liver can be similarly evaluated under ad lib with those fed by moderate (65%) FR. Male rats were given Phenobarbital (100 mg/kg) or Clofibrate (500 mg/kg) daily for approximately 14 weeks under ad lib or FR. The histomorphologic change in livers and hepatocellular BrdU labeling index were evaluated. The treatment-related histomorphologic change in liver included centrilobular hypertrophy in Phenobarbital group and diffuse hypertrophy and bile duct hyperplasia in Clofibrate group. The severity and incidence of these findings were comparable between ad lib and FR groups. The increase in hepatocellular labeling index was detected in the Phenobarbital-treated and Clofibrate-treated groups under both ad lib and FR even there were some quantitative differences in responses. In conclusion, effects on liver including hepatocellular proliferation activity of Phenobarbital and Clofibrate were detected under FR feeding as were detected in ad lib feeding.
5. Diuron and Mice Skin Carcinogenesis
Bianca Ferrucio1,2, Carla Adriene Franchi1,2, Maria Luiza Cotrim de Oliveira1,2, Deilson Elgui de Oliveira1,3, João Lauro Viana de Camargo1,2
1São Paulo State University (UNESP) - Botucatu Medical School, Deparment of Pathology, Botucatu, SP, Brazil, 2TOXICAM Laboratory, Botucatu, SP, Brazil, 3Molecular Pathology Laboratory, Botucatu, SP, Brazil
Diuron [3-(3,4-dichlorophenyl)-1,1-dimethyl urea] is a herbicide with carcinogenic activity in rodents. It has been categorized as “likely human carcinogen” by USEPA. Since it has been used in agricultural crops, it is necessary to clarify the mode of action (MOA) through which this herbicide induces rodent neoplasia and to understand the relevance of this MOA to humans. The initiating activity of Diuron was registered in an initiation-promotion mice skin carcinogenesis model, although several other reports have questioned its genotoxic potential. Two studies were developed in order to verify the initiating and promoting potentials of Diuron in mice skin. In the first one, Diuron 250 mg/kg b.w. diluted in dimethyl sulphoxide (DMSO) was applied on the mice interscapular region followed by 3,5μg/100μL 12-O-tetradecanoil phorbol 13-acetate (TPA), a well established skin promoting agent. A different group was topically initiated with 52μg/100μL 9,10-dimethyl-1,2-benzantracene (DMBA) followed by applications of Diuron/DMSO. Only the positive control group (DMBA+TPA) developed a significant incidence of papillomas. Since the possibility exists that DMSO could have significantly inhibited the skin response to a possible herbicide carcinogenic activity, a second study was developed using acetone as an alternative solvent. Again, only the control group developed papillomas, indicating that varying the solvents did not influence the results. Under the experimental conditions adopted, Diuron did not exert carcinogenic activity in the mice skin, in contrast with results described in the literature.
6. Naturally Occurring Bladder Papilloma and Lymphocytic Leukemia in an Aging Male Wistar Rat
Chien-Chao Chiu1, Hsiao-Li Chuang2, Yen-Te Huang1, Kinji Shirota3, Hans Hsien-Chuan Chen4
1National Laboratory Animal Center, National Applied Research Laboratories, Taipei, Taiwan, 2Department of Veterinary Medicine, National Chung Hsing University, Taichung, Taiwan, 3Research Institute of Biosciences, Azabu University, Sagamihara, Japan, 4HansChen BiotechPath Consulting, Taipei, Taiwan
Abstract: A 27-month-old male Wistar rat used in breeding purpose was submitted for euthanasia because of weight loss. At necropsy, splenomegaly (7.8 x1.9 x1.2 cm), hepatomegaly, and a large white-tan nodular mass (1.1 x 1.1 x 0.6 cm) superficially attached and merging in urine of the markedly dilated urinary bladder were the most significant gross observations. Microscopically, the spleen was replaced entirely by proliferative and infiltrating large type lymphocyte neoplastic cells but atrophic white pull was discernable in some areas. The liver was infiltrated diffusely with the large numbers of lymphocytic leukemia cells in dilated capillaries and sinusoids, and so were the adrenals, kidneys, lung, heart, and other tissues including the bladder. No mass or nodular forming neoplastic infiltration was noted in the liver portal areas or other tissues, an indicative of acute leukemia. The bladder had a well-formed transitional cell papilloma that was composed of crowded, massive growth of long, frond-like papilla of well differentiated urothelial cells lined on central core of loose fibrovascular stalk. Occasional mitotic figures were seen in this well-differentiated papilloma. The bladder wall was distended and atrophic without any evidence of cystitis or formation of calculus nedus. In addition, an advanced stage chronic progressive nephropathy was noted but without evidence of occurring metastatic calcification except for the gastric wall musculature of the glandular stomach. This case appeared interesting as to the bearing of well formed, spontaneous bladder papilloma with concurrent acute leukemia in an aging rat on extended lifespan approximately 2 months older than those used in conventional 2-year chronic toxicity/carcinogenicity studies.
7. Effect Of Zoledronic Acid Therapy In An In Vivo Model Of Bone Invasive Feline Oral Squamous Cell Carcinoma
Chelsea Martin, Jillian Werbeck, Nandu Thudi, Lisa Lanigan, Tobie Wolfe, Ramiro Toribio, Thomas Rosol
The Ohio State University, Columbus, Ohio, United States
8. Dose-response Study Of Diuron [3-(3,4-dichlorophenyl)-1,1-dimethylurea]-Induced Toxicity of the Urinary Bladder in Male Wistar Rats
Shadia Ihlaseh, Ana Paula Cardoso, Mitscheli Da Rocha, Merielen Nascimento, Maria Luiza De Oliveira, João Lauro De Camargo
Sao Paulo State University, Botucatu Medical School, Department of Pathology, Botucatu, SP, Brazil
Incidence of urothelial alterations by SEM
1Number of animals: 10/group;
2Classes 1-5: increased severity of urothelial alterations (Cohen et al., 2007);
3Fisher exact test.
9. A novel pan-HDAC inhibitor, OSU-HDAC42 exhibits biological effects against canine and human malignant mast cells via histone and Kit dependent pathways
Tzuyin Lin, Sridhar Murahari, Samuel Kulp, Dasheng Wang, Ching-Shih Chen, William Kisseberth, Cheryl London
Ohio State University, Columbus, OH, USA
Epigenetic changes including DNA hypermethylation and histone hypoacetylation are found in many cancers, and evidence suggests that inhibition of histone deacetylation is a promising approach to modulating epigenetic changes. Preliminary data indicate that histone deacetylase inhibitors (HDACis) may have biologic activity against canine malignant mast cell disease; however, the mechanisms remain unclear. The purpose of the studies was to investigate the histone and Kit-dependent mechanisms of pan-HDAC inhibition in malignant mast cells. Briefly, malignant mast cell lines with various forms of constitutively active Kit, normal canine bone marrow-derived mast cells, and primary canine malignant mast cells (n=17) were treated with OSU-HDAC42 (a novel phenylbutyrate-based HDACi) and 17-AAG, and cell viability, cell cycling, cell invasion, and effects on several signalling pathways were evaluated. Treatment of the malignant mast cells with OSU-HDAC42 induced growth inhibition, cell cycle arrest, apoptosis, and activation of caspase3/7 as well as invasion inhibition. OSU-HDAC42 also promoted hyperacetylation of H3, H4 and α-tubulin in addition to upregulation of p21. Downregulation of wild-type and mutant Kit occurred following OSU-HDAC42 treatment in a dose and time-dependent manner. Disassociation between Kit and HSP90 and upregulation of HSP70 were observed after OSU-HDAC42 treatment suggesting loss of HSP90 chaperone function. Additionally, OSU-HDAC42 downregulated pAkt, total Akt, pSTAT3/5 and total STAT3/5 and MMP9. In summary, these data are the first to provide evidence of Kit downregulation via an HSP90 dependent mechanism following HDACi treatment. The novel HDACi OSU-HDAC42 exhibits biologic anti-cancer effects representing a promising therapeutic approach for malignant mast cell disease.
10. Hepatocellular Carcinoma in Genetically Obese Mice
Hyojung Kwon1,2, Youngsuk Won1, Hyoungchin Kim1, Daeyong Kim2
1KRIBB, Chungbook, Korea, Republic of, 2Department of Veterinary Pathology, College of Veterinary Medicine, Seoul National University, Seoul, Republic of Korea
Various epidemiological studies have shown that obesity may be risk factor for hepatocellular carcinoma. However, underlying mechanisms of how obesity promotes hepatic carcinogenesis remain unknown. In this study, we investigated the susceptibility of genetically obese mice to diethylnitrosamine (DEN) induced hepatic carcinogenesis. The ob/ob, db/db and wild type (WT) mice were injected with a single dose of DEN at 12 days of age and killed at appropriate time points. Visible tumors developed as early as 16 weeks of age in genetically obese mice. Histological examination revealed that 100% of ob/ob and db/db mice developed hepatic adenoma and 100% of ob/ob and 30% of db/db mice developed hepatocellular carcinoma by 26 weeks, while no hepatic tumor was observed in WT mice. Phenotypically, body and relative liver weights, serum cholesterol, glucose and triglyceride levels of ob/ob and db/db mice were significantly higher than WT mice. Microarray analysis showed approximately 200 of the known genes was commonly expressed in both hepatic tumors of ob/ob and db/db mice, which were mostly related to the regulation of cell proliferation and translation. In conclusion, our data show that development of hepatic tumor is enhanced in genetically obese ob/ob and db/db mice. Futuremore, gene expression profiling of these mice models may provide novel insights into obesity-associated hepatic carcinogenesis.
11. Preclinical investigation of novel indole-3-carbinol analogs for the prevention and treatment of prostate cancer
Aaron Sargeant1, Chen-Hsun Tsai1, Alene Yeater2, Samuel Kulp1, Lisa Berman-Booty1, Robert Rengel1, Steven Clinton1, Dasheng Wang1, Ching-Shih Chen1
1The Ohio State University, Columbus, Ohio, United States, 2University of Toledo Medical Center, Toledo, Ohio, USA
We recently reported the anticancer activity of OSU-A9, a novel small-molecule agent which retains the broad-spectrum of antitumor activity of its parent compound indole-3-carbinol but with enhanced metabolic stability and two orders of magnitude greater potency in prostate cancer. Here, we assess both the efficacy and safety of orally-administered OSU-A9 vis-à-vis two slightly modified agents in a series of preclinical studies carried out, in part, in preparation for prevention and regression trials in transgenic mouse models of prostate cancer. Histopathology and body and organ weight evaluations of mice treated in a dose-ranging study showed that OSU-A9 was without toxicity up to 200 mg/kg/day. When administered daily to PC-3 xenograft-bearing nude mice at 25, 50, and 100 mg/kg, the drug suppressed tumor growth by 49%, 54%, and 55%, respectively, compared to vehicle-treated controls (P<0.05). Its fluorinated derivative OSU-A9F similarly suppressed tumor growth by 52%, 61%, and 68%, respectively, at these doses. Fifty mg/kg/day OSU-A9 prolonged the tumor-defined lifespan of a syngeneic model (C57BL/6 mice bearing subcutaneous TRAMP-C2 tumors) from 16 to 24 days (48% increase, P<0.01) in association with increased tumor apoptosis and decreased proliferation, whereas OSU-A9F increased the lifespan of this model by 32% at 50 mg/kg/day (P<0.05). Preliminary results of similar studies carried out with the methylated derivative OSU-A9M suggest a therapeutic advantage of this compound with greater potency. Transgenic mice receiving a drug-containing diet (planned for 450 ppm OSU-A9M) will be evaluated for the compound’s molecular and morphologic effects on the development of prostatic intraepithelial neoplasia and carcinoma.
12. Proteomic Profiling Of Multiple Myeloma Cells Following a High Dose Of Melphalan
Sheeno Thyparambil, Veronica MacLeod, John Shaughnessy, Ricky Edmondson
University of Arkansas for Medical Sciences, Little Rock, AR, United States
Introduction: Multiple myeloma (MM), plasma cell disorder, is the second most common hematological cancer. Melphalan, an alkylating agent, is a widely used chemotherapeutic agent in MM. In this study we used label-free high throughput proteomic technology-differential mass spectrometry (dMS), to identify proteins that were differentially expressed following a high dose of melphalan.
Methods: MM cells (ARK) were treated with 0, or 100 uM (high dose) melphalan for 24hrs. An aliquot of 100K live ARK cells (duplicate) were lysed and then digested using trypsin. The tryptic digest equivalent of 5000 cells (~1 ug) was analyzed in triplicate using a 2hr nanoLC/MS/MS workflow (LTQ-Orbitrap). Feature identification, extraction, and retention time alignment was conducted using the Elucidator system (Rosetta). A fold change cut-off of 1.5 with qvalue<0.001 were used for obtaining differentially expressed proteins. Pertinent pathways were studied Ingenuity
Results: A total of 2123 proteins were identified. Of these, 120 and 261 proteins were upregulated or downregulated respectively in the treated group. Top canonical pathways that were upregulated included oxidative phosphorylation, apoptosis etc. while the top downregulated pathways included ubiquitination and glycolysis pathways.
Conclusions: This is the first report of the use of dMS to identify protein and pathways that are differentially expressed in ARK cells following high dose of melphalan. Label-free dMS is a high throughput proteomic analysis technology that will enable us to deduce the pathways that needs to be followed by conventional toxicological and pathological techniques following dosing with any drug.
13. Stable Overexpression of DeltaNp63a Blocks Senescence and Promotes Malignant Transformation in Keratinocytes
Linan Ha, Roshini Ponnamperuma, Wendy Weinberg
DMA/OBP/OPS/FDA, Bethesda, MD, United States
The p53 homologue p63 is critical for squamous epithelial development, and elevated levels of the p63 isoform DNp63 are associated with squamous cell cancers of the head, neck, skin, lung and cervix. Our lab has shown that transient overexpression of adenoviral-DNp63a in primary mouse epidermal keratinocytes enhances cell proliferation and inhibits morphological and biochemical differentiation. Lentiviruses encoding DNp63a were developed to mimic the long-term over-expression observed in cancers, with an infection efficiency of >90%. Lenti-DNp63a expression was stable for >15 days, as seen by western blots and immunostaining. The S-phase population was consistently higher in lenti-DNp63a cultures vs. lenti-GFP control cultures (18.18%, 11.12% and 5.12% at days 5, 10, and 15, vs.17.58%, 4.12% and 1.59%). Keratinocytes expressing lenti-GFP, but not lenti-DNp63a, underwent spontaneous senescence by day 15, as evidenced by the expression of SA-b-gal or the presence of nuclear foci of heterochromatin protein 1gamma. Lenti-DNp63a also blocked oncogene-induced senescence typically seen within 15 days following introduction of v-ras-Ha. In in vivo animal studies, lenti-GFP and lenti-DNp63a keratinocytes consistently formed normal skin following grafting to nude mice (15 grafted mice/group). Lenti-GFP keratinocytes expressing v-ras-Ha developed benign well-differentiated papillomas in 4/15 grafted mice. In contrast, lenti-DNp63a/v-ras-Ha keratinocytes formed undifferentiated carcinomas in 20/20 mice. The average volume of lenti-DNp63a/v-ras-Ha tumors was 831.9mm3, compared to 25mm3 in the lenti-GFP/v-ras-Ha group. Our findings suggest that long-term overexpression of DNp63a promotes keratinocyte proliferation and inhibits senescence, thereby facilitating keratinocyte transformation.
14. Zoledronic Acid inhibits Osteolysis in a Nude Mouse Model of Canine Prostate Cancer Mixed Bone Metastases
Nanda Thudi, Chelsea Martin, Prasad Nadella, Soledad Fernandez, Jillian Werbeck, Joseph Pinzone, Thomas Rosol
The Ohio State University, Columbus, OH, United States
Bone metastasis is the most common cause of morbidity and mortality in patients with advanced prostate cancer and is manifested primarily as mixed osteoblastic and osteolytic lesions. However, the mechanisms responsible for bone metastases in prostate cancer are not clearly understood due to the lack of relevant in vivo models that mimic the clinical presentation of the disease in humans. We previously established a nude mouse model with mixed bone metastases using intracardiac injection of canine prostate cancer cells (Ace-1). In this study, we hypothesized that tumor-induced osteolysis promoted the incidence of bone metastases and osteoblastic activity.
We studied the effect of inhibition of osteolysis with zoledronic acid (ZA) on the prevention and progression of Ace-1 bone metastases in nude mice using prophylactic and delayed treatment protocols. Bioluminescent imaging, radiography, and histopathological evaluation were performed to monitor the effect of ZA on the incidence, progression and nature of bone metastases.
Unexpectedly, there was no significant difference in tumor burden and the incidence of metastasis between control and treatment groups as detected by bioluminescent imaging and bone histomorphometry. However, radiographic and histopathological analysis showed a significant treatment-related decrease in osteolysis, but there was no effect on tumor-induced trabecular bone thickness in both treatment groups compared to controls.
Our results demonstrated that the incidence of prostate cancer bone metastases in vivo was independent of tumor-induced osteoclastic bone resorption, even though inhibition of bone resorption reduced bone loss associated with the mixed osteoblastic/osteolytic bone metastases in the Ace-1 model.
15. A High Dose of Genistein Induces Cell Death in Uterine Leiomyoma Cells by Autophagy and not by Apoptosis
Lysandra Castro, Linda Yu, Alicia B. Moore, Grace E. Kissling, Retha R. Newbold, Darlene Dixon
National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina, United States
Previously, we have shown that a high dose of genistein induces apoptosis in uterine smooth muscle cells (UtSMC), but not uterine leiomyoma (UtLM) cells. To determine the mechanism(s) of genistein-induced cell death in UtLM cells versus UtSMC, and to better understand the regulation of UtLM cell death, mediators of apoptosis, cytotoxicity and autophagy were analyzed following exposure to genistein (50 μg/ml) at 72 h and 168 h. Results showed that the ratio of apoptosis mediators, bcl-2:bak, was significantly (p<0.05) increased in genistein-treated UtLM cells at 72 h, and by 168 h the ratio was still increased, but not significantly. Genistein decreased bcl-2:bak in UtSMC at 72 h, but by 168 h the ratio was significantly (p<0.05) increased. Expression of Fas ligand (Fas-L) and Fas receptor was enhanced in genistein-treated UtSMC compared to UtLM cells at both time points as evidenced by confocal microscopy; whereas, in genistein-treated UtLM cells the autophagy/autophagosome marker, MAPL1C3A, was overexpressed at 72 h, and by 168 h there was significant (p<0.05) cytotoxicity as shown by a membrane integrity assay. In conclusion, it appears that genistein induces overexpression of bcl-2, inhibits apoptosis, and allows the activation of an autophagy cell death pathway, which may lead to necrosis in genistein-treated UtLM cells; whereas, in UtSMC, genistein activates the extrinsic (Fas-L and Fas) apoptotic pathway. These data will help to define the biological mechanisms involved in the inhibition of UtLM cell growth, and possibly provide novel targets for clinical intervention for fibroids.
16. Identification Of Unique Chromosomal Alterations In Primary and Metastatic Pancreatic Ductal Tumors Derived From Transgenic Mice By Array-Comparative Genomic Hybridization (aCGH)
Melissa Schutten, Adam Jochem, Eric Sandgren
University of Wisconsin-Madison, Madison, WI, United States
Pancreatic ductal adenocarcinoma (PDA) is a devastating disease that affects 1 in 10,00 people in the United States today. PDA has a grave prognosis due, in part, to the neoplasm’s inherent resistance to current chemotherapeutic and radiation treatments. Understanding the molecular events underlying tumorigenesis and identifying the cell(s) of origin in this disease could direct the development of novel drugs that may impact clinical prognosis. Studies using human pancreatic tumor samples have identified several altered genes in PDA, such as mutations in the oncogene K-ras, overexpression of growth factors EGFR/Erbb2, and mutational inactivation of the tumor suppressor genes p53 and p16. By specifically targeting mutant K-ras to pancreatic acinar cells, in combination with partial loss of p16, our lab has developed a mouse model of PDA that accurately models the sequence of preinvasive ductal lesions and invasive adenocarcinoma. Using primary pancreatic tumors and paired liver metastases from these transgenic mice, we identified common regions of chromosomal amplifications and loss using array-Comparative Genomic Hybridization (aCGH). Among 30 primary tumors evaluated, areas of common chromosomal amplifications affected genes involved in extracellular matrix interactions (Adam 11/12), cellular proliferation (Hras) and cell cycle (dynactin). Common areas of chromosomal loss involved the tumor suppressor gene p16. Areas of chromosomal alterations unique to the paired liver metastases involved serine threonine kinases (Brsk3, Mapk1), growth factor signaling (Nrg3), and tumor suppressor function (Cdkn2b). Our studies identify genes unique to primary and metastatic pancreatic tumors that may serve as putative targets for drug development or clinical biomarkers.
17. Role of Eukaryotic Initiation Factor 4E (eIF4E) in Osteosarcoma Metastasis
Tanasa Osborne1,2, Ling Ren1, Wanda Haschek-Hock2, Stephen Hewitt3, Chand Khanna1
1Tumor and Metastasis Biology Section, Pediatric Oncology Branch, National Cancer Institute, Bethesda, MD, United States, 2Department of Pathobiology, University of Illinois at Urbana-Champaign, Urbana, IL, United States, 3Tissue Array Research Program (TARP), Rockville, MD, United States
The most significant problem for cancer patients is the development of metastatic disease. Emblematic of the problem is the clinical progression seen in most patients with osteosarcoma, where metastasis to the lung is the most common cause of death. Improvements in patient outcome require a better understanding of the biology of metastasis. Cancer cells are believed to efficiently regulate protein translation at specific times and locations in a cell in response to changes in their environment. Preventing the dynamic regulation of these proteins (many of which have been associated with cancer) may be an effective treatment strategy in the management of metastasis. Within the process of protein translation the abundance and activation of the mRNA cap-binding phosphoprotein, eukaryotic initiation factor 4E (eIF4E) is considered to be both rate and process limiting. To define the biological role of eIF4E in the metastatic phenotype of osteosarcoma we have employed a comparative approach to study the biology of metastasis in osteosarcoma by using tissues and reagents from murine and human osteosarcomas. Recently, we confirmed high and consistent expression of eIF4E in human osteosarcoma tissues and demonstrated our ability to detect and overexpress eIF4E protein in osteosarcoma cell lines of several species. We propose to modulate eIF4E expression in murine and human osteosarcoma cell lines using overexpression and knockdown techniques to define the role of eIF4E at various steps of the metastatic cascade in vitro and in vivo.
18. Evaluation of automated histology pattern recognition tool for selection and analysis of tumor regions in breast sections stained with ER, PR and Ki67
Kate Lillard-Wetherell
Aperio, Vista, CA, United States
Advances in genomic and proteomic technology have generated many candidate biomarkers for tumorigenesis & cancer progression. In oncology, immunohistochemical biomarkers are being developed as companion diagnostics. For breast cancer, a number of IHC biomarkers are analyzed routinely, including HER2, ER, PR, p53 and Ki67. Image analysis is often a bottleneck in IHC biomarker development, particularly in cancer research. Automated image analysis of tumors is complicated by the presence of both tumor and non-tumor cells (primarily stroma and infiltrating immune cells). The objective of this study was to evaluate the ability of a histology pattern recognition algorithm (Genie) to automate the isolation of tumor tissue from IHC-stained sections for subsequent image analysis.
A series of breast tissue sections stained for nuclear biomarkers, ER, PR, and Ki67, were scanned using an Aperio ScanScope instrument and analyzed using a Genie classifier algorithm to select tumor tissues. These results were compared to selection of neoplastic regions using manual annotation.
For ER, PR and Ki67 stained slides, the percentage of positively stained cells varied by 0.9%, 0%, and 5.6%, respectively between computer and manual scoring. For all three biomarkers, the overall intensity score was unaffected by the method of tissue selection. These results suggest that automated tumor tissue classification using histology pattern recognition may be a viable & time-saving alternative to manual tissue annotation for evaluating biomarker expression in neoplasms. These methods may be applied to facilitate the analysis of large biomarker panels, speeding the identification of the most promising biomarkers for further development.
19. Impact of Post Translational Modifications on the Function of of HTLV-1 p30
Nadine Bowden, Rajaneesh Anupam, Matt Kesic, Michael Lairmore
The Ohio State University, Columbus, OH, United States
Results: Interestingly, our radiolabeling methods detected a truncated version of p30; a single ~20 kD band. The expected 30 kD band was detected by western blot assay.
Discussion
Our results suggest that p30 is a phosphoprotein and is cleaved into 2 distinct forms. A 20 kd cleavage product is phosphorylated, opening the possibility that p30 functions are dependent on phosphorylation and dependent on 2 forms of the protein. By improving our understanding of the role of phosphorylation, and possibly cleavage in the functions of p30 we may reveal potential viral specific drug and or therapeutic targets.
20. Development Of An In-Vivo Bioluminescent Mouse Model of Prostate Cancer Metastasis
Philip Martin, Rachel Pierce, Kathleen Kelly
National Institutes of Health (NCI), Bethesda, Maryland, United States
Our goal is to develop a mouse model of prostate cancer metastasis that can be used to test mechanistic hypotheses regarding which signaling pathways drive prostate cancer metastasis. A transgenic mouse was bred with prostate specific probasin driven Cre deletion of Pten and P53 and constitutive luciferase expression to allow for in-vivo bioluminescent imaging. The tumorigenic and metastatic potential of the donor tumor cells were tested by orthotopic transplantation into immunocompromised mice. Donor mice develop prostatic intra-epithelial neoplasia (PIN) as early as 8 weeks, adenocarcinoma by 12 weeks, and death due to urinary outflow obstruction by 7.5 months. The adenocarcinoma is heterogeneous with the presence of CK8(luminal)+/CK5(basal)-, CK8+/CK5+, and CK8-/CK5+ populations in varying amounts, with occasional synaptophysin+ cells. In older mice there is evidence of epithelial-to-mesenchymal transition (EMT) as adenocarcinomas are admixed with spindle cell tumors with heterogeneous and reduced CK8. The tumors are highly invasive with occasional micrometastases in regional lymph nodes.. Orthotopic tumors were highly invasive adenocarcimonas, adenocarcinomas with a minor spindle cell component, basal cell/squamous carcinomas, or occasionally adenosquamous carcinomas. Often tumors had regions containing only CK8+ luminal cells, closely resembling human prostate adenocarcinoma. Metastases were not detected in primary recipient mice however secondary transplantation into beige-nude-Xid mice resulted in lung metastasis as early as 8 weeks post injection. Cell lines derived from these tumors will serve as platforms for future studies investigating the cellular origin and molecular pathological characteristics of prostate cancer metastasis.
22. Localization of ALT Isozymes in Beagle Dogs: An Immunohistochemical Study
Yuichi Takai, Yusuke Sudo, Kazuko Hirai, Masami Aoki, Ryo Fukuda, Kenji Takami
Takeda Pharmaceutical Company Limited, Osaka, Japan
Alanine aminotransferase (ALT) is a biomarker that widely used to assess hepatic damage in clinical and pre-clinical studies. Recently, two ALT isozymes (ALT1 and ALT2), that are coded on different chromosomes and expressed at different levels in various organs, have been found in humans, mice and rats [h1](Per Lindblom, et al.,2007; Sanjay B. Jadaho, et al., 2004); however these have not yet been elucidated in dogs. In the present study, we established specific rabbit polyclonal antibodies that recognize ALT1 and ALT2 in beagle dogs and immunohistochemically investigated the expression and distribution of these ALT isozymes in dogs.
ALT1 reactivity was found in the hepatocytes, bile ducts, glomeruli, proximal and distal renal tubules, renal pelvis, myocardiocytes, striated muscle, parietal cells, adrenal cortex and medulla and in the acinar and islet cells in the pancreas, while ALT2 was expressed in the bile ducts, proximal renal tubules, macula densa, renal pelvis, myocardiocytes, striated muscle, all cell types of the fundic glands, adrenal cortex and medulla, and in the acinar and islet cells in the pancreas. As with these enzymes in the other species, in dogs the ALT isozymes are not co-localized in some organs. In addition, localization of these isozymes in the liver, kidney, heart, stomach, pancreas, and kidney in dogs was different from that in humans. The present findings are considered to be important in the interpretation of elevated ALT levels, and the establishment of analytical methods for the serum/plasma ALT isozymes is expected to provide a more comprehensive understanding of the significance of increased ALT levels, especially when increased ALT levels without co-existing histopathological changes indicating hepatocellular injury are noted in toxicity studies.
23. Spontaneous Glomerulonephritis in Four Göttingen Minipigs
Enrico Vezzali1, Rosa Anna Manno1, Yuval Ramot2, Abraham Nyska3,4
1R.T.C. – Research Toxicology Centre S.p.A., Pomezia, Rome, Italy, 2Hadassah – Hebrew University Medical Center, Jerusalem, Israel, 3Consultant in Toxicological Pathology, Timrat, Israel, 4Sackler School of Medicine, Tel Aviv University, Tel Aviv, Israel
The Göttingen minipig is commonly used in the safety assessment of drugs as a non-rodent species recommended by various regulatory authorities for preclinical studies. However, spontaneous pathology in this species has rarely been described. The knowledge of spontaneous renal lesions might have a significant impact on the interpretation of any drug-induced pathology during preclinical testing. The present study focuses on the renal pathology background in Göttingen minipigs used in preclinical studies at RTC. Four out of 157 microbiologically defined Göttingen minipigs, 4 to 10 months old and weighing 9 to 22 kg, supplied by Ellegaard Göttingen Minipigs (Dalmose, Denmark) from the end of 2007 to the beginning of 2009, showed asthenia or poor health conditions, leading to humane sacrifice. Grossly, the kidneys appeared enlarged and/or with dark red discolouration. Histopathological evaluation revealed moderate to severe renal lesions in the four animals, characterised by membranous or membranoproliferative glomerulonephritis at different stages, accompanied by secondary tubulo-interstitial involvement. The renal changes observed were suggested to be spontaneous in origin. In conclusion, 2.5% of the examined minipigs presented glomerulonephritis that, to the best of our knowledge, has rarely been signalled in Göttingen minipigs as background pathology.
24. Atypical Cellular Infiltrates In Mouse And Rat Liver Following Treatment With A Pan-caspase Inhibitor
Kathryn Gropp1, Daniel Morton1, Frances Clemo1, John Kreeger1, Bernard LeBlanc2, Thomas Kawabata1, Daniel Lettiere1, Kathleen White3, William Brown3, Tim Coskran1
1Pfizer, Inc., Groton, CT, United States, 2Pfizer, Inc., Amboise, France, 3Pfizer, Inc., Kalamazoo, MI, United States
A pan-caspase inhibitor, with both anti-apoptotic and anti-inflammatory responses, produced atypical cellular infiltrates (ACI) in livers of CD-1 and hemizygous rasH2 transgenic mice and Sprague-Dawley rats. ACI consisted of large and small lymphocytes, large lymphoblasts, macrophages and occasional eosinophils and were often associated with larger hepatic blood vessels. In mice, ACI were present in liver after 1 month of either oral or drug-in-diet dosing. Following 3 months of drug-in-diet dosing, ACI in liver occurred at all doses tested with increasing incidence, size, and severity at higher doses. ACI was also present in kidney and adrenal gland. ACI at higher doses developed follicle-like structures which labeled strongly with antibodies to the B-cell antigen CD45/B220. Lymphoblasts within ACI were positive for T-cell antigen CD3 and/or CD45/B220, and ACI contained many Ki67 positive cells. There were no histomorphologic findings in the surrounding hepatic parenchyma at any dose tested. ACI were not fully reversible in mice after a 2-month recovery period. During a 3-month drug-in-diet rat study, smaller ACI were present in only livers in 3/7 rats which died early (days 30-78). The immunohistochemical staining profile of ACI in rats was similar to that in mice. In conclusion, atypical cellular infiltrates were observed in liver of mice and rats and were probably a result of disruption of normal lymphocyte homeostasis by pan-caspase inhibition of apoptosis and other cellular functions.
25. Histology Atlas of the Developing Mouse Hepatobiliary System
Laura Wilding1, Julie Foley2, Susan Elmore2
1North Carolina State College of Veterinary Medicine, Raleigh, NC, United States, 2National Institute of Environmental Health Sciences, Cellular and Molecular Pathology Branch, Research Triangle Park, NC, United States
Congenital defects of the hepatobiliary system are recognized in animals and humans and are poorly understood. Animal model phenotyping, in-utero exposure toxicity studies, and investigation into causes of embryonic or perinatal deaths have led to increased requests for pathologists to diagnose congenital developmental disorders. In mammals, the liver is the largest internal organ and it performs many critical functions – it is the main site of hematopoiesis during fetal development and has both endocrine and exocrine functions important for maintaining homeostasis. Due to its significant role, alterations in the size, morphology, or function of the liver often lead to embryonic lethality. Many publications describe individual aspects of hepatobiliary development at defined stages; however, no single resource provides a detailed histological evaluation of H & E stained sections of the developing liver and biliary system. As such, the work herein describes the creation of a histology atlas of hepatobiliary development which contains high-magnification and high-resolution color images illustrating the normal architecture and histology of the developing liver and biliary system from embryonic day (e) 9.5-18.5. This atlas includes images and descriptions of normal microanatomy of the developing liver, as well as descriptions of changes in the individual cell populations and in cellular organization that occur as the liver matures. Although the focus of this work is normal hepatobiliary development, common defects in liver development are also described to serve as a reference for pathologists who may be asked to evaluate and recognize fetal and postnatal mice with congenital hepatobiliary defects.
26. Degenerative Renal Tubular Lesions in F344N Rats Exposed to β-Myrcene
Mark F. Cesta2, Robert C. Sills2, Po Chan1, Gordon C. Hard3
1Toxicology Operations Branch, NTP, NIEHS, NIH, DHHS, Research Triangle Park, NC, United States, 2Cellular and Molecular Pathology Branch, NTP, NIEHS, NIH, DHHS, Research Triangle Park, NC, United States, 3Private Consultant, Tairua, New Zealand
β-Myrcene, an acyclic, unsubstituted monoterpene used widely in cosmetics, soaps, detergents, and as a flavoring additive in food and beverages, was tested in gavage studies on male and female rats by the NTP. Groups of 10 rats received 0, 0.25, 0.5, 1, or 2 g/kg β-myrcene for 3 months and groups of 50 received 0, 0.25, 0.5, or 1 g/kg for 2 years. In the subchronic and chronic studies, there were dose-related increases in the incidence and severity of renal tubule nephrosis and exacerbation of chronic progressive nephropathy in both sexes. Nephrosis primarily affected the outer stripe of the outer medulla and comprised the following lesions: renal tubule dilation, tubular epithelial karyomegaly and hyperplasia, tubular atrophy, hyperplasia of the collecting duct epithelium, and interstitial fibrosis. In the subchronic study, there was a dose-related increase in hyaline droplet accumulation (consistent with α2u-globulin nephropathy) in the 0.25 and 0.5 g/kg male dose groups, which then decreased to zero in the 2.0 g/kg group. In the 2-year study, there was a treatment-related increase in the incidence of linear papillary mineralization, a chronic manifestation of α2u-globulin nephropathy, in the 0.25 and 0.5 g/kg male dose groups, but not the 1.0 g/kg group. Renal tubule nephrosis has not been seen in previous NTP studies. α2u-globulin nephropathy, chronic progressive nephropathy, and renal tubule nephrosis are degenerative diseases of renal tubules that result in increased cell turnover. The contribution of each of these diseases to the treatment-related renal neoplasia seen in these rats remains uncertain.
27. Role of CYP1C1 and CYP1A Expression in PAH Induced Fundulus Hepatic Lesions
Lu Wang1, Alvin Camus2, Cammi Thornton1, Wu Dong1, Kristine Willett1
1University of Mississippi, University, MS, United States, 2University of Georgia, Athens, GA, United States
CYP1C1 is a relatively new member of the cytochrome P450 family 1 found in teleost. Amino acid analysis suggests CYP1C1 may structurally and/or functionally similar to mammalian CYP1B1 which has been recognized for its role in tumorgenesis. We have hypothesized that CYP1C1 genes may be involved in the molecular mechanisms of PAH carcinogenesis. Fundulus fry were exposed at 6-8 days post-hatch (dph) and again at 13-15 dph for 6 hr to control, 5 mg/L benzo[a]pyrene, and 5 mg/L dimethylbenzanthracene. Both CYP1A and CYP1C1 protein expression was induced within 6 hr and immunohistochemistry (IHC) results suggested strong CYP1C1 expression in kidney and GI. Eight months after initial exposure, gross liver lesions were observed during dissection in 20% of the BaP and 35% of the DMBA-treated group. Histopathologic findings included the presence of cellular alteration (eosinophilic and basophilic foci), hepatocellular adenoma, hepatocellular carcinoma and billiary neoplasm. Inflammatory lesions including lymphocyte inflammation and hepatic granulomas with fibrosis tissue were also observed. IHC was performed with CYP1A and CYP1C1 antibodies. Strong CYP1A signal was detected in cellular foci across the hepatocyte cytoplasm and in hepatocelluar carcinomas, intense CYP1A signal was detected at the border of the tumor (invading cancer cells) while a lower amount was seen in the rest of the neoplasm. In contrast, CYP1C1 was only detectable and highly expressed in proliferating bile duct epithelial cells. Our results suggest a important role of CYP1C as well as CYP1A in carcinogen bioactivation and tumor formation. This project is supported by NIEHS (R01ES012710).
28. Uterine Trophoblastic Tumors in Macaques
Sunish Mohanan1, Jennifer Cann1, Hermina Borgerink1, Justin D. Vidal2, Ross P. Tarara3, J. Mark Cline1
1Department of Pathology/Comparative Medicine, Wake Forest University School of Medicine, Winston Salem, NC, United States, 2GlaxoSmithKline, King of Prussia, PA, United States, 3California National Primate Research Center, UC Davis, Davis, CA, United States
Trophoblastic disease in humans includes various types of malignant neoplasms (choriocarcinoma, placental site trophoblastic tumor and epithelioid trophoblastic tumor), two benign entities (exaggerated placental site and placental site nodule) and malformations of the chorionic villi (hydatidiform mole). The malignant tumors usually follow a normal or ectopic pregnancy or abortion. Nonhuman primates also develop trophoblastic diseases but few cases are reported. We present the histologic and immunohistochemical features of trophoblastic tumors in three adult female, uniparous or multiparous, macaques. All three animals presented with abnormal chronic vaginal bleeding and anemia. The tumors were confined to uterus and in one case the tumor was detected in the uterine stump, eight years after hysterectomy. Histologically the neoplasms were composed of moderately to densely cellular sheets and nests of uni- and multinucleated oval to polygonal cells supported by fine fibrovascular stroma which expanded the endometrium to varying degrees and in one case invaded the myometrium. The neoplastic cells were 20-50um in greatest dimension with abundant eosinophilic cytoplasm and large vesicular nuclei with prominent nucleoli. Anisocytosis and anisokaryosis were marked and mitotic figures were rare. Immunohistochemistry revealed diffuse positive cytoplasmic staining for pan-cytokeratin in all neoplastic cells; vimentin staining was negative. 10-40% of the neoplastic cells, including most multinucleated cells, show moderate to intense cytoplasmic staining for human chorionic gonadotropin. Multifocally, 5-30% of the neoplastic cells showed faint to moderately intense cytoplasmic staining for human placental lactogen. These rare but distinctive neoplasms have not been recognized previously in the uterus of macaques.
29. Influence Of Diuron On The Rat Urinary Bladder Mucosa: An Urinary Acidification Study
Mitscheli Da Rocha, Merielen Nascimento, Ana Paula Cardoso, Ednéia Zelandi, João Lauro De Camargo, Maria Luiza De Oliveira
São Paulo State University - UNESP, Botucatu Medical School, Department of Pathology, TOXICAM laboratory, Botucatu/São Paulo, Brazil
In a long-term study, high dietary concentration of Diuron, a substituted urea herbicide, was carcinogenic to the urinary bladder of rats. The accepted non-genotoxic mode of action (MOA) of Diuron encompass urothelial cell exfoliation and necrosis induced by urinary precipitates, crystals, followed by regenerative cell proliferation and sustained urothelial hyperplasia that may favor neoplasia development. The aim of this study was to evaluate the role of urinary acidification on Diuron-induced urothelial carcinogenesis. Male Wistar rats were allocated to nine groups fed with Diuron 2.500 ppm associated or not to NH4Cl 10.000 ppm for 15, 25 and 30 weeks. NH4Cl was used to acidify the urine. One group treated only with Diuron during 15 weeks was also fed the following 15 weeks a Diuron-free diet to evaluate the potential reversibility of urothelial lesions. At each moment of evaluation, both Diuron and Diuron+NH4Cl groups presented increased incidences (p<0.05) of simple urothelial hyperplasia, a putative preneoplastic lesion. Urinary acidification reduced the amount of crystals and precipitates. Increased incidences of urothelial lesions were also detected under scanning electron microscopy (SEM) in both groups at the 15th week, when compared to the control group. Therefore, urothelial lesions seem to be not related to the urinary sediment. At the end of the study, the reversibility group presented decreased incidence of lesions under light and SEM, not different from the control group. These data suggest that urinary acidification, precipitates and crystals did not play a role in the development of putative urothelial lesions induced by this herbicide.
31. Hepatotoxic Effect Of TCPOBOP Correlates With Constitutive Androstane Receptor Activation In Mice
Heidge Fukumasu1,3, Gabriel Anhé2, Daniel Soares Sanches1, Leonila Ester Reinert-Raspantini1, Silvana Bordin2, Jerrold M. Ward4, Maria Lucia Dagli1
1Faculty of Veterinary Medicine and Animal Sciences - University of Sao Paulo, Sao Paulo, Sao Paulo, Brazil, 2Institute of Biomedical Sciences - University of Sao Paulo, Sao Paulo, Sao Paulo, Brazil, 3Faculty of Animal Science and Food Engineering - University of Sao Paulo, Pirassununga, Sao Paulo, Brazil, 4Global VetPathology, Montgomery Village, Maryland, United States
The Constitutive Androstane Receptor (CAR) has been recently identified as a key regulator of drug metabolism genes mediating the induction of several phase I and II enzymes, as well as transporters. The pesticide contaminant 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP) is considered the most potent CAR ligand in mice, where cyp2B10 is one of its targets. Herbal components present in diet or from complementary and alternative medicine (CAM) could interact with these receptors and this can mechanistically occur at both transcriptional and post-transcriptional levels. Also, many of these CAM induced hepatotoxic effects in animals and humans but so far no mechanism of action is known. So, the aim here was to determinate if the level of CAR transcriptional activation might be correlated with liver damage in mice. Female C57BL/6 mice were administered once with TCPOBOP by gavage (3, 6 or 9 mg/kg). Twenty-four hours later, they were euthanized, necropsied, blood sera were collected for ALT analysis and livers were collected for histology and Chromatin immunoprecipitation assay (ChiP) for CAR transcriptional activation. The histological analysis revealed mild liver vacuolar degeneration after 24h, which increases with the dose of TCPOBOP. In addition, dose-dependent effects of TCPOBOP were noted in increased liver weight (p<0.0001), ALT levels (p=0.0002) and transcriptional activation of cyp2B10 by CAR (p=0.0399). Spearman’s non-parametric correlation analysis showed that ALT levels correlated positively with CAR transcriptional activation (p=0.0068).These results lead us to initiate studies to examine involvement of death pathways with CAR activation. Special attention is given to the role of Mdm2 transcriptional activation by CAR.
32. A Possible Implication of Thioredoxin in Liver Regeneration
Byung-Il Yoon1,3, Mi-Sun Park2, Yong-Baek Kim3, Jeong-Hee Han1, Dae-Yong Kim4
1School of Veterinary Medicine, Kangwon National University, Chuncheon, Gangwon-do, Korea, Republic of, 2National Cancer Center, Goyang, Kyunggi-do, Korea, Republic of, 3College of Veterinary Medicine, North Carolina State University, Raleigh, NC, United States, 4College of Veterinary Medicine, Seoul National University, Seoul, Korea, Republic of
Liver is a unique organ in which mature hepatocytes reconstitute the injured liver with the ability of self-renewal and proliferation. The scheme of liver regeneration (LR) is strictly controlled by a number of genes regulating cell proliferation and apoptosis. Thioredoxin (Trx) functions as a cell proliferating promoter and an anti-apoptotic factor in cells. In the present study, we investigated the role of Trx in LR after 1/3 or 2/3 partial hepatectomy (PH), using Trx transgenic (Tg) and hetero-knockout mice (hKO) as well as wild-type mice (WT). In addition to the measurements of liver volume, protein level of cell proliferating nuclear antigen (PCNA), and Trx reductase (TrxR) activity at multiple time points of LR, we performed time-based quantitative analyses of DNA synthesis and Trx mRNA during LR in the mice with different genetic background. Cell proliferating activity was gradually increased and highest at 3 days after PH. Significant increase of TrxR activity was consistently noted in the mouse livers with 2/3 PH throughout the LR. Trx mRNA level dynamically oscillated depending on the phase of LR. DNA synthesis of liver cell components was significantly enhanced in the Trx Tg mice compared to that in the hKO or WT mice. Our results strongly support that Trx plays a critical role in the LR process, possibly associated with cell proliferation and/or cell apoptosis regulation.
33. Metabolomics Analysis of Urine and Kidney Tissue in Rats Treated with Valproate
Kurt Boudonck, Kirk Beebe
Metabolon, Research Triangle Park, NC, United States
Valproate (VPA) is a drug widely used to treat epilepsy, but it has serious adverse effects including hepatotoxicity, teratogenicity and antifolate activity. Rats were treated with VPA (500 mg/kg) or saline (vehicle) once daily for 1, 5 or 28 days. Urine and kidney tissue samples were collected from groups of 6 rats each (12 rats for vehicle group) at days 1, 5 and 28. Full data curation of kidney tissue samples yielded 547 metabolites, and urine yielded 657 metabolites, consisting of amino acid metabolites, peptides, carbohydrates, lipids, energy metabolites, nucleotides, cofactors, vitamins and xenobiotics.
34. Repeated Dose Oral Toxicity Study of Febuxostat In Sprague Dawley Rats
Praful Patel, Vijay Patel, K.S. Prajapati, Rajesh Sunder, Mukul Jain, Pankaj Patel
College of Veterinary Science and Animal Husbandry, Anand Agricultural University, Anand , Gujart, India
35. Kidney Changes After Daily Slow-bolus IV Injection of 10 mL/kg Cremophor EL® in 5% Dextrose for 2 Weeks to Wistar Rats
Kathryn Bowenkamp, Karen Killary, Danielle Diaz, Donna Raab, Gregory Argentieri, Raviprakash Dugyala
Novartis Institutes for BioMedical Resaerch, East Hanover, NJ, United States
Polyoxyl 35 castor oil USP (a.k.a. Polyoxyethylenglyceroltriricioleat 35 (DAG); Cremophor ELÒ) is a non-ionic solubilizer and emulsifier used for aqueous preparations of hydrophobic substances in pharmaceuticals, cosmetics and feedstuffs. Cremophor EL is soluble in water and ethanol, and the degree of solubilization achieved depends on the amount of Cremophor EL used. Oral, topical, and inhaled delivery of Cremophor EL is well tolerated in many species. Percutaneous Cremophor EL also is reported to be well tolerated in rats but may cause anaphylactoid reactions in cattle and humans. In a recent study, sterile-filtered 95% Cremophor EL (65/35 v/v) in 5% Dextrose (1:10 ratio) was the vehicle for once daily intravenous injection into the tail vein of Wistar rats for 2 weeks at volumes of 2 or 10 mL/kg BW. Rats receiving 10 mL/kg Cremophor EL became pale and ataxic with decreased locomotor activity. In males body weight gain and food consumption decreased. No clinical pathology findings were attributable to the kidney. At necropsy kidney weights were increased in rats that received 10 mL/kg Cremophor EL vehicle, and segmental vacuolation of proximal tubular epithelium was seen. Vacuole contents were unlabeled by Oil-Red-O staining for neutral triglycerides or lipids. Ultrastructural examination of revealed increased numbers of distended lysosomes containing variably electron-dense granular to acicular material. Renal tubular vacuolation following daily slow IV bolus injection of 10 mL/kg Cremophor EL vehicle was considered secondary to lysosomal accumulation of excess vehicle or metabolites in young-adult Wistar rats.
ÒCremophor EL is a registered trademark of BASF Aktiengesellschaft.
37. Blockade Of Adenosine A1 Receptors With BG9928 Prevents Cisplatin-Induced Acute Renal Injury In Rats
Wendell Davis1, Eric Whalley1, Barry Ticho1, Kathleen Wortham1, Allan Gill2, Don Costab3
1Biogen Idec, Cambridge, MA, United States, 2ALS Therapy Development Institute, Cambridge, MA, United States, 3EnVivo Pharma, Watertown, MA, United States
Cisplatin, an anti-tumor agent induces nephrotoxicity and renal failure due to renal vasoconstriction and decreased glomerular filtration, changes believed to involve accumulation of adenosine and activation of adenosine A1 receptors (A1AR). This study investigated the effect of an orally active A1AR antagonist, BG9928 versus furosemide on biochemical measures of renal function and histopathology in cisplatin-induced acute renal injury in rats. BG9928 (1mg/kg bid on days 0-1 or 0-6) and furosemide (30 mg/kg bid p.o. on days 0-5) were administered to groups of female Sprague-Dawley rats pre-treated with cisplatin (5 mg/kg i.v.) or vehicle. Cisplatin treatment alone produced marked renal injury with elevations in creatinine (3.7 +/- 0.41 mg/dL) and BUN (146.2+/- 18.3 mg/dL), significant reductions in body weight, and maximal renal pathology scores. BG9928 administered from days 0-1 or 0-6 resulted in significant reductions in creatinine and BUN, accelerated the return of these biochemical indices and body weight to baseline levels, and improved renal histopathology scores. Administration of furosemide resulted in significantly elevated BUN (5.7 +/-1.3 mg/dL) and creatinine (207 +/-52 mg/dL) levels, reduced body weight, and demise of all animals by day 7. These data suggest that administration of the A1AR antagonist BG9928 ameliorated the effects of cisplatin-induced acute renal injury in rats. Moreover, the A1AR antagonist BG9928 may be useful in the treatment of renal failure induced by such agents as cisplatin and in other conditions such as impaired kidney function in heart failure patients where activation of A1AR’s by locally produced adenosine in the kidney is known to occur.
38. Nonneoplastic Eye Lesions in Rats and Mice
Margarita Gruebbel1, Mark Hoenerhoff2
1Experimental Pathology Laboratories, Research Triangle Park, United States, 2National Institute of Environmental Health Sciences, Research Triangle Park, United States
Nonneoplastic lesions of the eye occur with variable incidences in acute and long-term toxicity studies in rats and mice. In many studies, incidental ocular findings are noted in control as well as treated animals. Such findings are attributable to various causes unrelated to test article including trauma, environmental (husbandry) conditions, bacterial and viral infections, develop-mental malformations, genetic predisposition, metabolic diseases, and aging changes. This presentation illustrates several incidental nonneoplastic eye lesions in rats and mice, using representative examples from F344 rats (albino) and B6C3F1 mice (pigmented) from 2-year carcinogenicity bioassays, 90-day subchronic toxicity studies, and lifespan studies conducted at the National Toxicology Program (NTP). The lesions depicted include inflammatory, degenerative, hyperplastic and dysplastic changes of the cornea, anterior segment, lens, uveal tract, vitreous, retina, sclera, bulbar conjunctiva, and retrobulbar tissue.
39. Spontaneous Aortitis in the Balb/c Mouse
Rossa Anna Manno1, Yuval Ramot2, Yoshimasa Okazaki3, Vezzali Enrico1, Michael Krakovsky4, Itschak Lamensdorf4, Moran Meiron5, Amir Toren5, Efrat Zehavi-Goldstein5, Abraham Nyska6
1RTC S.p.A., Pomezia, Rome, Italy, 2Hadassah– Hebrew University Medical Center, Jerusalem, Israel, 3Astellas Pharma Inc., Toxicologic Pathology, Drug Safety Research Laboratories, Osaka, Japan, 4Pharmaseed Ltd., Ness-Ziona, Israel, 5Pluristem Therapeutics Inc., Haifa, Israel, 6Sackler School of Medicine, Tel Aviv University, Timrat, Israel
Vasculitis in animal models is induced experimentally and occurs spontaneously. To evaluate vascular toxicity in drug safety studies, investigators must know background ranges, especially for the aortic base, which, due to its unique location, poses a diagnostic challenge. We examined whether high incidence rates (18-56%) of inflammation in the root of the aorta detected in a Balb/c mouse model for hind limb ischemia were related to the surgical procedure. Twenty mice underwent ligation of the femoral artery; incidences of inflammation were compared to those in controls. We used a multiple-section sampling to increase diagnostic rates. Although a cumulative incidence of 12.5% was found, no difference occurred in the overall incidence rates between the two groups. Evaluation of blood levels of inflammatory cytokines showed that ligation of the femoral artery produced higher levels of interleukin-6, consistent with its known pro-angiogenic role. The development of spontaneous arteritis in this strain must be considered in future studies, particularly following its utilization in models for vascular lesion induction. The diagnostic rate of vasculitis at the root of the aorta was increased by using a “multiple-section sampling” approach. Therefore, to achieve an accurate assessment of vascular inflammation at the root of the aorta, because of its increased susceptibility, we recommend a step-section sampling for accurate diagnosis.
40. Doxorubicin Associated Dilated Cardiomyopathy in a German Shepherd Dog
Tiffany Reed1,2, Joshua Webster1,2, Stephen Lenz1,2, Shawna Greene3
1Purdue University Animal Disease Diagnostic Laboratory, West Lafayette, In, United States, 2Purdue University Department of Comparative Pathobiology, West Lafayette, IN, United States, 3Purdue University, School of Veterinary Medicine, West Lafayette, IN, United States
A 13.5-year-old, spayed female, German Shepherd dog was necropsied following a seven-month chemotherapeutic regimen with doxorubicin for hepatic hemangiosarcoma. On gross examination of the heart, the myocardium was diffusely pale tan with white streaks extending throughout the papillary muscles. Both cardiac ventricles were dilated. Left and right ventricular free wall thickness was 1.5 cm and 0.8 cm, respectively. The pericardial sac contained 20 ml of red, translucent fluid. Histologically, myocardial fibers were thin and wavy, consistent with dilated cardiomyopathy. Many myocytes were distended by degenerative vacuoles, a classic finding in doxorubicin toxicosis. Hemangiosarcoma was still present in the liver. Other histologic lesions were chronic interstitial nephritis, necrotizing enteritis, and pulmonary interstitial fibrosis. Doxorubicin is cytotoxic via inhibition of DNA-synthesis, DNA-dependent RNA synthesis, and protein synthesis. Chemotherapy with the antineoplastic doxorubicin may be warranted; however, the side-effects may outweigh the benefits. Prolonged treatment can culminate in cardiotoxicosis, bone marrow suppression, nephrotoxicosis, vomiting, and diarrhea. This case highlights the effects of cumulative doxorubicin treatment in dogs and rationalizes further investigation of doxorubicin cardiotoxicosis as a model to develop new cardiotherapeutic drugs.
41. Mycoplasma Associated Arthritis And Weight Loss In Xenograft Bearing SCID-Beige Mice
Michael Linn, Laura Singer, Brian Higgins, Gary Greenstein, Kenneth Kolinsky, Wei He, Kathryn Packman
Hoffmann-La Roche, Nutley, NJ, United States
During the in-life portion of two efficacy studies, severe combined immunodeficienct beige (SCID-bg) mice, implanted with PANC-1- or SKOV-3-human xenograft tumors, developed swollen hocks and/or unexplained weight loss. Tissue samples were submitted for microbiology and histopathology evaluations. Aerobic cultures were negative. Histopathology revealed destruction of the joints with dense infiltration by neutrophils and macrophages, osteolysis, and fibrosis. No evidence of neoplasia was observed. A hock sample from a PANC-1 implanted mouse and a stock vial of SKOV-3 cells tested positive for Mycoplasma sp. by PCR. Further ELISA testing revealed the SKOV-3 cells were positive for M. orale. PANC-1 cells tested negative for Mycoplasma sp. The infected SKOV-3 cell line was considered the source of mycoplasmal infection, which cross-contaminated the PANC-1 cells during the cell culture scale-up process prior to implantation, and resulted in clinical signs in SCID-beige mice implanted by either xenograft tumor model.
42. Comparison of the Acute Effects of Inhaled Sulfur Mustard and its Surrogate, CEES, in F344 Rats
Thomas March, Janet Benson, Gary Grotendorst, JeanClare Seagrave, Waylon Weber
Lovelace Respiratory Research Institute, Albuquerque, New Mexico, United States
The chemical warfare agent, sulfur mustard (SM), has had a recent resurgence in deployment and is a potential terrorists’ weapon. SM causes skin blisters, corneal injury, and an ARDS-like pulmonary injury. Because of safety concerns and legal restrictions on SM possession, many laboratories use “half-mustard” surrogates, including 2-chloroethyl-ethylsulfide (CEES), to study effects of mustards. Few direct comparisons of SM and CEES have been made, and no comparison of the inhaled mustards has been reported. Here, ethanolic solutions of the mustards (1% w/v for SM and CEES; 5% for CEES, only) were nebulized and delivered by nose-only inhalation to female F344 rats for 5 – 15 minutes. Aerosol concentrations were ~200 μg/L (1% solutions) and 1.2 mg/L (5% solution). Rats were killed 1-3 days post-exposure; organs were collected for histopathology; lungs were lavaged for cytology and chemistry; and blood was analyzed for hematology and cytokines. Minimal changes were obtained with 1% CEES. Both SM and 5% CEES caused similar severity of necrosis and inflammation in the upper respiratory tract, but 5% CEES caused more severe pulmonary injury than SM. Both SM and 5% CEES caused increases in lung lavage inflammatory cells, increases in lavage and serum pro-inflammatory cytokines, epithelial apoptosis in the gastrointestinal tract, hemoconcentration, and lymphopenia. Only SM caused panleukopenia and hypoplasia of myeloid and erythroid bone marrow cells. Because of the qualitatively similar respiratory tract toxicity but different hematologic changes, CEES may be an adequate surrogate for assessing acute pulmonary, but not systemic, effects of inhaled SM.
Funded by U54 NS058185-02, NIH /NINDS.
43. Ocular Toxicity Caused by Paclitaxel in Neonatal Sprague-Dawley Rats
Maki Kuwata, Katsuhiko Yoshizawa, Miyo Matsumura, Kanji Takahashi, Airo Tsubura
Kansai Medical University, Moriguchu, Japan
Introduction: The toxic effects of paclitaxel (PTX) on neonatal eyes have not been evaluated. Methods: PTX was dissolved in solvent containing polyethoxylated castor oil and intraperitoneally administered to male and female Sprague-Dawley rats at a dose of 0, 2, 4, and 8 mg/kg at 0 day of age, 4 mg/kg at 14 days of age, or 8 mg/kg at 12-18 weeks of age. Eyes were histologically examined 1 and/or 7 days after PTX. Results: Male and female rats that received 4 mg/kg or more of PTX at 0 days of age developed cataracts and retinal dysplasias, while the rats that received other dosing regimens did not develop ocular lesions. Epithelial cells in the lens were apoptotic on day 1, and lens fibers were degenerative at day 7, indicating the development of cataracts. We observed scattered foci of apoptosis in the neuroblastic layer of the retina on day 1, and rosettes were seen on day 7, suggestive of retinal dysplasia. Discussion: Neonatal rats that received a threshold dose of PTX (4 mg/kg) at a critical period (0 days of age) developed cataracts and retinal dysplasia; however, the 2 mg/kg dose at 0 days of age or the 4 or 8 mg/kg dose at 14 days of age or older caused no ocular damage. Thus, the determination of the dose and timing of PTX treatment administered during the early developmental stage requires great care to avoid ocular toxicity.
44. A Model of Sulfur Mustard Induced Skin Injury in the Hairless Guinea Pig: Time Course of Lesion Development and Drug Efficacy Evaluations
Thomas March1, Janet Benson1, JeanClare Seagrave1, Waylon Weber1, Gary Grotendorst1, Gregory Schultz2
1Lovelace Respiratory Research Institute, Albuquerque, NM, United States, 2College of Medicine, University of Florida, Gainesville, FL, United States
The objective of these studies was to develop an animal model of skin injury induced by the chemical warfare agent, sulfur mustard (SM), for assessment of pathogenesis and efficacy of new therapeutics. Skin lesions in hairless guinea pigs were produced from neat SM deposited on filter paper inside Teflon cups adhered to the backs of the animals. Vapor concentrations achieved in the cups were ~400 μg/L. Exposure duration determined the dose, and 6 and 12 minutes of exposure produced sufficient lesions. In time-course studies, SM-exposed animals were killed from 6 hours to 14 days post-exposure. The efficacy of topical nanodoxycycline gel (Dox) to reduce lesion severity was also assessed. Endpoints included evaluations of erythema/edema, histopathology, and biochemical analysis of matrix metalloproteinase (MMP) activity. The MMP-2 and -9 activities were increased in both the 6- and 12-minute dose groups by 24 hours post exposure but remained elevated through 2 weeks only in the high-dose group. The low dose induced dermatitis; basal cell apoptosis /necrosis, hydropic degeneration, and spongiosis; and, at the later time points, acanthosis and hyperkeratosis. Lesions were more severe in the high dose group. Dox, as a MMP inhibitor, slightly decreased some of the degenerative and necrotizing changes evident in the low dose-exposed skin sites at 24 hours. Dox was ineffective at treating the more severe necrotizing and degenerative changes associated with high-dose SM exposure at 24 hours but slightly decreased the inflammation, edema, and hyperplasia evident at 96 hours. Inhibition of MMP activity may prove efficacious in treating SM-induced skin injury.
Funded by NIH NINDS #5U54NS058185-03.
45. The Cynodon dactylon Extract Help Improve Anticholinesterase Effects and Oxidative Stress Caused by Carbofuran in Rat Brain
Devendra K. Rai1, Prashant K. Rai2, Geeta Watal2 and Bechan Sharma1
1Department of biochemistry, University of Allahabad; Allahabad, India: 211 0022Department of Chemistry, University of Allahabad; Allahabad, India: 211 002
47. Methotrexate-induced Temporal Transcriptomic Changes in the Rat Duodenal Mucosa
Yi Yang, Rita Ciurlionis, Wayne Buck, Eric Blomme
Abbott Laboratories, Abbott Park, IL, United States
Gastrointestinal toxicity is one of the most common side effects of cytotoxic drugs such as methotrexate. The purpose of this study was to evaluate intestinal gene expression changes in a rat model of methotrexate-induced intestinal damage. Male Sprague-Dawley rats were administered 2.5 mg/kg methotrexate subcutaneousely for 1 or 3 consecutive days before necropsy. Two additional groups of rats were sacrificed after a recovery period of 3 and 5 days after 3 days of exposure to methotrexate. In addition to standard histopathological evaluation, rat duodenal mucosa was collected to evaluate the temporal transcriptomic changes in the course of intestinal degeneration and recovery. The results showed down-regulation of enterocyte-specific marker genes on day 3 of treatment and day 3 of recovery. There were minimal changes in the expression levels of marker genes for Goblet and Paneth cells. Genes involved in the nutrient metabolism pathways were down-regulated on day 3 of treatment and day 3 of recovery. Up-regulation of genes involved in cell cycle control and down-regulation of genes involved in DNA repair, cell cycle arrest, and apoptosis were noted on day 3 of recovery. Gene expression patterns were returned to baseline by day 5 of recovery. These results provide a further understanding of the molecular events governing to the chemotherapeutic agent-induced intestinal damage and its repair process.
49. A Bilateral Dystrophic Calcinosis Circumscripta in a Cynomolgus Macaque (Macaca fascicularis)
Kenichi Sato1, Zaher A. Radi2, Norbert Makori1, Dale Morris2, James Klaassen1, Steven Meyer1, Ryoichi Nagata3
1SNBL USA Ltd., Everett, WA, United States, 2Pfizer Global R&D, Chesterfield, MO, United States, 3Shin Nippon Biomedical Laboratories, Ltd., Tokyo, Japan
A well-circumscribed, bilateral, expansile, and nonencapsulated footpad neoplasms and missing digits were detected in a 6-year-old, female cynomolgus macaque from a routine toxicology study. These footpad neoplasms were composed of variably sized and firm nodules containing white chalky-like material in the subcutaneous tissue on cross section. Microscopically, the neoplasms were composed of cystic structures containing amorphous to granular, light to dark, basophilic materials that were surrounded by macrophages, multinucleated giant cells and fibrous connective tissue. By histochemistry, the neoplasm contents were von Kossa and Alizarin red S positive. Serum calcium and serum inorganic phosphorus levels of this monkey were within normal ranges. Based on the gross pathology, histopathology, serum chemistry, and histochemistry, a diagnosis of dystrophic calcinosis circumscripta was made. Dystrophic calcinosis circumscripta is an uncommon syndrome of mineralization characterized by deposition of calcium salts in soft tissues. Dystrophic calcinosis generally occurs following tissue damage while idiopathic calcinosis occurs in normal soft tissues with no abnormalities of calcium and phosphorus homeostasis. To our knowledge, this is the first report of dystrophic calcinosis circumscripta in cynomolgus macaque.
50. Assessment of the Cardiotoxicity of Hydralazine Using an Ultrasensitive Flow-based Troponin Immunoassay
Igor Mikaelian1, Denise Coluccio1, Gerard Hirkaler1, John C. Downing1, Erik Rasmussen1, John Todd2, Joel Estis2, Quynh Ahn Lu2, Rosemary Nicklaus1
1Hoffmann-La Roche, Inc., Nutley, NJ, United States, 2Singulex, Inc., Hayward, CA, United States
The purpose of this study was to correlate histopathology to serum troponin I (cTnI) assayed with the Erenna™ Immunoassay System in Wistar rats (Crl:Wi(Han)) using the hydralazine model of cardiotoxicity. A single dose of hydralazine caused an increase of cTnI at 6 hr post dose, followed by a sharp decrease at 24 hr and a return to baseline values at 48 hr. The second dose of hydralazine caused a smaller magnitude of cTnI increase at 6 hr than the first dose and a return to baseline values at 24 hr. The elevated values of cTnI coincided with acute myocardial necrosis at histology. However, elevated cTnI in the absence of histopathologic lesions was identified in multiple rats. As cTnI levels decreased, histopathologic changes in the heart matured to cardiomyophagy. In conclusion, cTnI measured by the Erenna™ Immunoassay System was more sensitive than histopathology to identify acute cardiotoxicity. However, the window of cTnI elevation was short, and only histopathology detected the damage at 24-48 hr after the episode of acute myocardial necrosis.
51. Kainate-induced Myocardial Necrosis and Coronary Arteritis Were Mediated by Different Pathway
Kiyokazu Ozaki, Tetsuro Matsuura, Isao Narama
Setsunan University, Hirakata/Osaka, Japan
Kainate (KA) is one of the neurotoxic analogs of excitatory amino acids that induce acute neuronal death in hippocampus in rodent animals. In addition, we have previously reported KA induces coronary arteritis and focal myocardial necrosis in mouse heart. In this study, we investigated heart damage was mediated by either brain activation or direct vascular effects of kainate.
In vitro experiment: Response of isolated heart to kainate was examined using the Magnus method.
In vivo experiment: Five-week-old male ddY mice were treated with phentolamine (PH), alpha-adrenergic antagonist, at 0 (control) and30 mg/kg intravenously twice. 12.5 mg/kg of kainate were treated intravenously 20 min after the second dosing of PH. Brains and hearts of surviving mice were examined 48 hours after dosing.
In vitro study, heart rate was significantly decreased with KA treatment. In vivo study, within one hour after KA treatment, 36% of control mouse died immediately after the appearance of seizure. PH pretreatment decreased mortality to 8%. Coronary arteritis was induced 45% of control and 21% of PH pretreated mice. Myocardial necrosis was observed 25% of control, and none of PH pretreated mice. Neuronal death of hippocampus was induced in 32% of control mice and none of PH pretreated mice. PH pretreatment perfectly inhibited neuronal death. Neuronal death was significantly correlated with myocardial necrosis, but unrelated with coronary arteritis.?
These data suggest that two different pathways to induce myocardial necrosis and coronary arteritis, because the incidence of the lesions has strong correlation with existence of brain lesions and sizure.
52. Summary of Laryngeal Lesions in Fischer 344 Rats and B6C3F1 Mice in National Toxicology Program (NTP) Chronic Inhalation Studies
Glen Marrs1, Rodney Miller1, Ronald Herbert2
1Experimental Pathology Laboratories, Inc., Research Triangle Park, NC, United States, 2National Institute of Environmental Health Sciences, Research Triangle Park, NC, United States
A comprehensive review summarizing data of laryngeal lesions recorded in NTP studies would be of value to serve as a reference for future microscopic evaluations of rat and mouse larynges. Most irritant-based laryngeal lesions observed would be expected to be applicable to any rat or mouse strain.
Forty-eight technical reports of peer-reviewed, finalized, chronic (two-year) inhalation studies conducted by the NTP from its origin through February, 2009 were reviewed. The recorded microscopic findings in the larynges were summarized.
Incidences of primary neoplasms, non-primary neoplasms and non-neoplastic lesions recorded within the larynges of control or treated, male or female, Fischer 344 rats and B6C3F1 mice are presented. Selected spontaneous and induced laryngeal lesions include squamous metaplasia, necrosis, inflammation, mineralization, hyperplasia, ulcer, degeneration and inflammatory polyp. The base of the epiglottis was the most common site affected and, thus, was considered the most sensitive site. Induced lesions were observed anterior and posterior to the base of the epiglottis, but never to the exclusion of the base of the epiglottis.
One of the more commonly diagnosed test-material-induced lesions of the larynx was squamous metaplasia. Often, in studies in which squamous metaplasia occurs in the in the larynges of rats or mice, it is asked if progression to neoplasia is possible. In the reviewed NTP studies, when squamous metaplasia was a test-material-induced effect, there was no indication that laryngeal neoplasia was treatment related.
53. Temporal Changes in Bone Marrow, Thymus and Lymph Nodes in Rhesus Macaques Following Total Body Ionizing Radiation Exposure
Mantena Srinivasa R1, Bartholomew, Amelia2, Lublimov, Alexander2, Lindeblad, Matthew O2, Walling, Brent E1, Haschek, Wanda M1
1University of Illinois at Urbana-Champaign, IL, USA. 2University of Illinois at Chicago, IL, USA.
Radiation exposure induces lymphoid, hematopoietic, gastrointestinal and pulmonary toxicity. Hematopoetic and lymphoid systems were evaluated in a pilot study to define the dose response curve of total body ionizing radiation. 33 adult male rhesus macaques, Macaca mulatto, were exposed to 675 (n=4), 720 (3), 755 (5), 785 (8), 805 (5) or 840 (8) Gy and received medical intervention. 27 died or were euthanized moribund within 59 days and 6 were euthanized at study termination, approximately 60 days. Bone marrow, lymph node and thymus were evaluated histologically. Bone marrow changes were correlated with hematologic data. By 22 days post exposure there was severe lymphoid depletion with diffuse histiocytic infiltration of the thymic cortex and medulla and sinuses of the lymph nodes; bone marrow was markedly hypocelluar (< 1%). Bone marrow cellularity and the number of megakaryocytes (per 4x field) in animals with unplanned necropsy were markedly decreased (11% and 3, respectively), compared to animals surviving to study termination (73% and 50). Bone marrow changes were independent of radiation dose but strongly correlated with survival time. WBC, RBC, and platelets markedly decreased by 22 days (0.1675x103, 2.89x106 and 15x103 /μL, respectively) as compared to study initiation (4.91x103, 4.53x106 and 308x103/μL) while less marked changes were seen in animals surviving greater than 22 days (8.96x103, 4.41x106 and 229x103 /μL) from study initiation (5.48x103, 4.68x106 and 315x103/μL). Lymphoid and hematopoetic systems returned to near normal in animals that survived to study termination. Medical intervention that included blood transfusion could have obscured dose response to radiation treatment. Both the bone marrow and hematologic changes were independent of radiation dose.
54. Focal Myocardial Degeneration Induced by Phenylhydrazine in the Rats
Hiroshi Edamoto, Chiharu Komiya, Masahiro Mochizuki, Etsuko Ideno, Daichi Nakamura, Yuko Ichikawa, Yoshito Nishihara, Kazutoshi Tamura
Bozo Research Center Inc., Gotemba, Japan
Phenylhydrazine hydrochloride (PHZ) is well known to induce hemolytic anemia. However there are no available reports on cardiac toxicities. The aim of the present study was to examine the myocardial damages after PHZ treatment.
Thirty Sprague-Dawley rats of each sex were injected intraperitoneally with 80 mg/kg of PHZ, and sacrificed at 8, 24 or 72 hours after treatment (HAT). Control animals were injected intraperitoneally with physiological saline.
After the treatment with PHZ, the skin rapidly changed black in color, suggesting methemoglobinemia, and it persisted until 24 HAT. Hematologically, red blood cell count, hemoglobin concentration, and hematocrit ratio were gradually decreased at all point examined, and red blood cell count were markedly decreased at 72 HAT. In contrast, methemoglobin concentration was increased at all points examined, and peaked at 8 HAT. Blood biochemically, plasm AST, LDH, and CPK levels began to elevate at 8 HAT, reached peak at 24 HAT, and then returned to almost the control level. Histopathologically, at 8 HAT, a few small foci of myocardial necrosis with mononuclear cell infiltration occurred predominantly adjacent to the endocardium in the left ventricle. At 24 HAT, such lesions were slightly increased in number, and were observed widely distributed throughout of the both side of the ventricles. At 72 HAT, no lesions were observed in any animals. The present study demonstrates that a single high-dose injection of PHZ can induce myocardial damage.
55. Background Cardiomyopathy in Cynomolgus Monkeys (Macaca fascicularis)
Tanja S. Zabka, Mudher Albassam
Roche, Nutley NJ, United States
A previously undescribed background cardiomyopathy was identified by routine light microscopic examination of the heart from three clinically healthy purpose-bred cynomolgus monkeys that ranged from four to nine years of age and included 2 males and 1 female. Special stains of Sirius red, Masson’s trichrome, and Mallory’s phosphotungstic acid hematoxylin (PTAH), and immunohistochemistry using anti-CD68, troponin-I and desmin were used to facilitate lesion characterization and assess cardiomyocyte viability. Microscopically, the cardiomyopathy had a consistent morphology and distribution. The apical to mid-ventricular myocardium to subendocardium had foci of cardiomyocyte disarray, vacuolization of the perimyseal connective tissue, and a meshwork of fibrous tissue surrounding medium-sized blood vessels and dissecting between or less often replacing cardiomyocytes. Foci had a minimal, predominantly macrophage infiltrate. The disrupted cardiomyocytes were immunoreactive to desmin and troponin-I antibodies and had a normal cross striation pattern by PTAH, indicating the chronic cardiomyopathy was not associated with active cardiomyocyte damage. The consistent morphology and distribution of the cardiomyopathy suggested a common etiology and pathogenesis and had features reminiscent of chronic catecholamine-induced experimental cardiomyopathy and stress cardiomyopathy in monkeys and humans, respectively. This report documents another background spontaneous heart lesion in clinically healthy monkeys for consideration during interpretation of toxicology studies.
56. Background Inflammatory Changes within the Stomach of Cynomolgus Monkeys (Macaca fascicularis): Influence of Geographical Origin and Age
Nicholas Macri, Creasy Dianne
Huntingdon Life Sciences, East Millstone, New Jersey, United States
Cynomolgus monkeys (Macaca fascicularis) are the most common non-human primate used for preclinical safety assessment. Inflammatory lesions of the stomach mucosa are frequently seen as background findings and can be a confounding factor for identifying target organ toxicity in this species. The purpose of this study was to characterize microscopic changes in the stomachs of vehicle-treated cynomolgus monkeys necropsied over a 5-year period. H&E stained sections from the cardia/fundus, body, and pylorus/antrum were evaluated in 206 animals (98 males and 108 females) ranging in age from 2 to 9.5 years and originating from Mainland Southeast Asia, and the islands of Indonesia and Mauritius. The most significant microscopic findings were the presence of epithelial changes associated with mucosal inflammatory cell infiltrates in the mucosa of the body of the stomach. The changes were characterized by cytoplasmic basophilia and decreased mucin content of the surface epithelial cells, increased epithelial mitoses in the underlying neck region of the gastric pits and decreased numbers of parietal cells. The inflammatory infiltrate was predominantly lymphocytic, with occasional minimal acute inflammation within gastric glands. Inflammation and epithelial changes were more common in animals originating from Mainland Southeast Asia (25%) than Indonesia (8.6%) or Mauritius (0%) and had an inverse relationship between severity and age. Other factors such as vehicle administration and route of administration showed no correlation with the gastric inflammation. The histopathologic findings identified in this study are consistent with those described for H. pylori infection in Cynomolgus monkeys originating from the Philippines.
57. Cadmium Induces Maladaptive Cardiac Hypertrophy in Pregnancy
Kathleen Gabrielson, Djahida Bedja, Polina Sysa, Xin Guo, Yi Xu
The Johns Hopkins University, Baltimore, MD, United States
Cardiac hypertrophy, an increase in ventricular mass, is due to an increase in cardiomyocyte size. Hypertrophy can be adaptive, in response to enhanced hemodynamic loads, such as in pregnancy or maladpative, as seen in hypertension.. The causes of pregnancy hypertrophy that becomes maladaptive are not understood. Here, we investigate pregnancy-induced left ventricular hypertrophy and the role of cadmium, an environmental metal in water, tobacco smoke, road dust and food. We hypothesized that cadmium would increase cardiac hypertrophy in pregnant mice.
58. Quantifying Huntingtin Protein Aggregates in a Transgenic Mouse Model of Huntington’s Disease
Kevin Liang, Trevor Johnson
Aperio, Vista, CA, United States
Huntington’s Disease (HD) is a progressive neurodegenerative disease associated with the intracellular and extracellular aggregation of the protein, Huntingtin (HTT), in the striatum and cortex of the affected brain. HD has been well studied due in large part to the development of transgenic rodent models. Historically, analyzing neurohistological preparations from a glass slide have been difficult from a quantification standpoint due to the size of tissue sections. However, digital whole-slide imaging systems allow researchers to quantitatively evaluate entire tissue sections, dramatically enhancing the speed and accuracy of such research studies. In this report, we quantify and compare HTT protein aggregate density within the striatum and the cortex of coronal brain slices.
Brain slices from Q140 Knock-in animals were immunostained with an anti-HTT antibody and scanned using the Aperio ScanScope. Digital images were analyzed using the ImageScope® Nuclear analysis tool.
The results show that HTT protein aggregation is more severe in the striatum compared to cortex for the same brain sections of the same transgenic animal. This is a significant finding, as it is clear that the physical symptoms of HD not only manifest sooner, but also more severely than the cognitive disorders associated with the disease. Future investigations should include a developmental time course of HTT protein aggregation in the brain that is correlated to motor and cognitive behavioral performance. While it will be important to segregate the differential aspects of the animals’ behavioral performance (motor impairments vs. cognitive impairments), obtaining an accurate quantitative assessment of histopathology is clearly achievable.
59. Cynomolgus Monkey Retina: Characterisation of Cell Types by ImmunohistoChemistry
Alessandro Piaia, Alberta Argentino Storino, Patrizia Cristofori
GlaxoSmithKline, Verona, Italy
The apparent simplicity of the layered structure of the mammalian retina is contrasted by the complexity of cell types, their interaction and the plethora of neurotransmitter receptors and receptor subunits in the different cell population. In particular, AMPA glutamate receptors are expressed by OFF-cone bipolar cells, rod bipolar cells, horizontal cells, amacrine cells, and ganglion cells, acting on the conducting pathway. In our work we set a robust and easily reproducible method to detect different population of cells in the retina of Macaca fascicularis by immunohistochemistry on Davidson-fixed eyes. We also investigate the detection of AMPA glutamate receptor subunits GluR1, GluR2, GluR3 or GluR4 by the same method. With this protocol, we found that Parvalbumin was a suitable marker for the identification of Horizontal cells, Calretinin for Amacrine cells; Protein kinase C alpha for Rod ON bipolar cells, Recoverin for the identification of OFF Cone Bipolar Cells in the monkey retina. GluR4 immunoreaction was positive in the photoreceptor layer, and in the OFF cone bipolar cells and horizontal cells layer, at the cone pedicle base. This protocol is considered useful for the early detection of histologic changes in the retina, in association with objective measures of retinal functional alteration, like multifocal electroretinography (mfERG) and /or electro-oculography (EOG). The research described in this paper complied with national legislation and with the company policy on the care and use of laboratory animals and with related codes of practice.
60. A Treated Urban Sewage Sludge Sample - In Vivo Toxicity and Genotoxicity Evaluations
Marize de Lourdes Marzo Solano1, João Francisco Lozano Luvizutto1, Paula Regina Pereira Silva1, Patrícia Lepage Alves de Lima1, Daniele Passarelli1, Gisela de Aragão Umbuzeiro2, João Lauro Viana de Camargo1
1Botucatu Medical School, UNESP - São Paulo State University, Botucatu, São Paulo, Brazil, 2Environmental Toxicology, Genotoxicity and Microbiology Division - CETESB, São Paulo, São Paulo, Brazil
Disposal of the sewage sludge produced by urban wastewater treatment plants is of worldwide concern. Because recycling and agricultural use has been proposed, the potential adverse health effects and risk to humans should be verified. The present study was conducted to determine in rats the potential oral toxicity and genotoxicity of sewage sludge samples obtained from a specific urban treatment plant that generates 7 tons of sludge/day from domestic, textile and dyeing industries effluents. Male and female Wistar rats were fed ad libitum during 13 weeks a pelleted diet containing 0, 5,000, 10,000 and 50,000 ppm of the treated sludge. Animals of each group were sacrificed on the 28th day of study. In these animals, and in the surviving animals sacrificed after 90 days of experiment, no alterations were registered on food and water consumptions, on body and organ weights and on blood cell counts, when compared to the respective controls. Histological alterations were also not observed in all collected organs of the high-dose treated animals. The potential mutagenic and the genotoxic effects were evaluated respectively by the bone marrow micronucleus test and by the comet assay with periorbital plexus blood cells. The present findings indicate the specific sludge sample studied does not induce systemic toxicity or genotoxicity when administered at very high doses during up to 90 days to both genders of Wistar rats. Even though the data is negative, this result provides important findings regarding the risks of exposure to treated sewage sludge.
61. 13-Week Repeated Dose Toxicity and Genotoxicity Studies of Cimicifuga heracleifolia Extract
Jeong-Hwan Che1, Ji-Ran You1, Yun-Soon Kim1, Seung-Hyun Kim1, Ja-June Jang2, Hee-Chan Kim1, Byeong-Cheol Kang1,2
1Clinical Research Institute, Seoul National University Hospital, Seoul, Korea, Republic of, 2Seoul National University College of Medicine, Seoul, Korea, Republic of
Cimicifuga heracleifolia (Cimicifuga racemosa, Black Cohosh) is commonly used for treatment of symptom relief during and after the menopause. Recently, adverse liver reaction associated with black cohosh was reported. However, repeated toxicity and genotoxicity studies for Cimicifuga heracleifolia extract (CHE) was not carried out yet. Therefore, this study was performed to evaluate 13-week repeated dose toxicity (13-RDT) and genotoxicity studies according to the NTP protocol and GLP regulation.
In preliminary study, CHE was not observed toxic response up to 2000 mg/kg B.W. in 14-day study. Based on preliminary results, CHE was orally administered five times per week for 13 weeks in F344/N rats at dosage levels of 0, 25, 74, 222, 667, and 2000 mg/kg/B.W. In 13-RDT study in male and female rats reported no significant adverse effects in mortality, clinical signs, body weight changes, food and water consumption, hematology and serum biochemistry parameters, male and female reproductive tissue evaluations, gross pathology and histopathology. Therefore, NOAEL for CHE might be thought 2000 mg/kg in males and females. CHE did not show any potential to induce gene mutations in reverse mutation tests using Salmonella typhimurium TA98, TA100, TA102, TA1535, and TA1537 strains. CHE did not induce chromosomal aberrations in cultured Chinese hamster lung cells. In a micronucleus assay in mice, CHE did not induce any micronuclei or suppress bone marrow, indicating it does not cause chromosome aberrations.
The results from these safety studies support the safety of CHE for human consumption.
62. Ozone Exacerbates Acetaminophen-Induced Lung AND Liver Toxicity In Mice
Daher Ibrahim Aibo, Neil Birmingham, Jane Maddox, Robert Roth, Patricia Ganey, James Wagner, Jack Harkema
Michigan State University, East Lansing, Michigan, United States
Though airway epithelium is the primary target tissue for toxicity caused by inhaled ozone (O3), changes in gene expression have also been reported in the livers of O3-exposed mice. An overdose of acetaminophen (APAP) is the most common cause of drug-induced liver injury in people. High doses of APAP also cause liver and lung toxicity in mice. The purpose of our study was to determine the effects of inhaled O3 on APAP-induced lung and liver toxicity. Mice were treated with APAP (300 mg/kg) or saline, and exposed 2h later to 0.5 ppm O3 or air for 6h. Mice were killed 9h or 32h after APAP treatment. At 32h, APAP caused necrosis and increased DNA synthesis in both airway epithelium and hepatic parenchyma. At the same time point, APAP/O3 mice had greater airway and liver necrosis with attenuated DNA synthesis compared to APAP/Air mice. At 9h, APAP/O3 mice had greater hepatic and pulmonary increases in gene expression of HO-1 (oxidative stress marker) and P21 (regulator of cell cycle progression) and greater pulmonary expression of IL6 (pro-inflammatory cytokine) compared to APAP/Air mice. Mechanisms underlying O3 exacerbation of APAP toxicity are yet unknown. These results, however, suggest that high ambient O3 may pose a health threat to subpopulations taking high therapeutic doses of APAP or suffering from lung or liver disease.
63. Phospholipidosis Timecourse in HepG2 Cells Treated with Amiodarone: Transmission Electron Microscopic and Gene Expression Analysis
Shannon Burroughs1,2, Mary Keener1, Connie Cummings1, Lawrence Yoon1, Richard Peterson1
1GlaxoSmithKline, Safety Assessment, Research Triangle Park, NC, United States, 2North Carolina State University, College of Veterinary Medicine, Raleigh, NC, United States
Phospholipidosis (PLD) is a common test article-related change in preclinical toxicology. Cationic-Amphiphilic Drugs (CADs) bind to and inhibit function of lysosomal phospholipase or bind to/inhibit degradation of phospholipids. Amiodarone, a CAD, has been shown clinically and preclinically to be phospholipidotic. HepG2 cells, a human hepatocarcinoma cell line, were treated with 25μM Amiodarone hydrochloride or vehicle, and cells were harvested at 0, 6, 24, and 48 hours post exposure. Cells were divided, one half was processed for transmission electron microscopy (TEM) and the other processed for total RNA isolation/gene expression analysis. TEM showed a lack of lysosomal multilamellar bodies (LMBs) in vehicle control cells at all timepoints and in treated cells at 0 hours, and a time dependent increase in the number, complexity, and size of LMBs, as well as increased lipid between 6 and 48 hours. TaqMan evaluation of transcript levels of genes chosen from the literature and internal data, was performed comparing treated and control cells at all timepoints. Changes in the following genes: >1.4 fold increase over controls [phenylalkylamine Ca2+ antagonist binding protein (Ebp; 24 hrs.), squalene epoxidase (sqle; 6-48 hrs.), acid ceramidase (ASAH1; 24 and 48 hrs.), inhibin beta E (InhbE; 24 and 48 hrs.), nuclear protein 1 (Nupr1; 24 and 48 hrs.), and hepsin (Hpn; 24 hrs.)], <0.7 fold decrease over controls [steroid sulfatase (Sts; 48 hrs.) and solute carrier family 2 (facilitated glucose transporter) member 3 (Slc2a3; 24 and 48 hrs.)] suggested potential biomarkers. The genes are associated with steroid biosynthesis, lipid metabolism/phospholipid degradation, cell cycle/proliferation/death, proteolysis, androgen/estrogen metabolism, and transport pathways, respectively. These data allowed temporal evaluation of the ultrastructure, as well as the gene expression fingerprint of PLD development in vitro. Further steps include in vivo validation of this gene expression profile with Amiodarone and other CADs.
64. Immunosuppressive Effects of Pteridium aquilinum In Mice Are Reversed by Selenium Supplementation
Andreia Oliveira Latorre, Beatriz Dorr Caniceiro, Heidge Fukumasu, Silvana Lima Górniak
University of São Paulo, São Paulo, SP, Brazil
Pteridium aquilinum (Pa) is a poisonous plant known as carcinogenic for animals and humans. Moreover, our earlier studies showed that Pa causes immunosuppressive effects, such as atrophy of white pulp of spleen and reduction on natural killer (NK) cell cytotoxicity. Therefore, here we evaluated if selenium (Se) administration could reverse or prevent these effects. For analysis of spleen damage 50 male C57BL/6 mice were separated into 5 groups as follows: Co, P, PSe, WP, and WSe, treated by gavage either water, Pa (30g/kg) or Se (1.3mg/kg) for 30 days. For NK cell cytotoxicity evaluation were prepared spleen cell suspensions from eight male C57BL/6 mice. Cultures for each treatment were performed as follows: untreated, Se 0.1mM, aqueous extract of Pa 4mg/ml (AE4), AE4Se (co-incubated), and AE4wSe (AE4/1hr wash and incubated with Se 0.1mM/1hr), incubated for 1hr or 2hr at 37°C in a humidified atmosphere with 5% CO2. For assay of NK cell cytotoxicity, these treated cells were washed, adjusted in complete RPMI, and used as effectors cells and the YAC-1 cells as target. After 4 hr of incubation at 37°C with 5% CO2, propidium iodide was added and the samples were acquired by flow cytometry. We observed a complete reversion of atrophy of white pulp of spleen with Se treatment in vivo. In addition, the study in vitro revealed prevention and reversion of reduction on NK cell cytotoxicity by Se treatment. Thus, we hypothesized that immunostimulation induced by Se treatment could effectively reverse the immunotoxic effects caused by Pa in mice.
65. Effects of Diphenyl Diselenide and Ebselen on Acidosis and Iron-induced Lipid Peroxidation in Rat Kidney Homogenates
Waseem Hassan, Joao B T Rocha
Departamento de Química, Centro de Ciências Naturais e Exatas, Universidade Federal de Santa Maria, Santa Maria, CEP 97105-900, RS, Brazil, Santa Maria -RS, Brazil
In this study we explored the relationship between lipid peroxidation and acidosis in kidney homogenate following incubation at different pH ranging from physiological to acidic values. Lipid peroxidation assessed by thiobarbituric acid-reactive substances (TBARS) production, was markedly enhanced as the solution pH was lowered from 7.8 to 5.4. The binding capacity for some iron-binding proteins is highly pH sensitive and the iron mobilized may come from reserves where it is weakly bound. The degree of Fe (II) - induced lipid peroxidation of the extract was also dependent on pH of the reaction mixture such that TBARS production increased when extract was incubated in the pH range of 5.4 – 6.8. Diphenyl diselenide significantly protected TBARS production at all studied pH values in a concentration dependent manner while ebselen offered only a small statistically non-significant protection. However, changing the pH of the reaction medium did not alter the anti-oxidant activity of the tested compounds. This study provides in-vitro evidence for acidosis and Fe (II) induced oxidative stress in kidney homogenate and anti-oxidant action of diphenyl diselenide.
66. Process Validation of Tissue Cross Reactivity Studies Using CD209 in Cynomolgus Monkey
Mylène Valin1, Thomas Lemarchand1, Bernard Palate1, Alexandre Fifre2, Roy Forster1
1CIT, Evreux, France, 2Inserm Paris XII Univ., Paris, France
Cross reactivity is the ability of an antibody to react with or bind to an antigen that did not stimulate its production. Tissue cross reactivity (TCR) studies are required for regulatory filing of monoclonal antibody therapeutics. The objective of the present study was to validate the techniques and procedures used in our laboratory for TCR studies. For this purpose we evaluated the cross reactivity of mouse IgG1 anti-human CD209 antibody in normal cynomolgus (Macaca fascicularis) tissues. CD209 (DC-SIGN) is a C-type lectin receptor present on both macrophages and dendritic cells. An isotype-matched control (mouse anti-rat CD8) was included in the study. Anti-CD209 was tested on a panel of snap frozen cynomolgus tissues (~40 organs) and in human liver and lymph nodes. Immunohistochemistry was performed with Discovery XT autostainer (Ventana) with a Dab Map procedure. The determination of optimal concentration of antibody was made on cynomolgus liver using Morpho-expert image analysis software (Explora Nova). Validation included precision, reproducibility and range/linearity. A semi-quantitative visual evaluation of staining was performed. Staining was observed in the majority of tissues, probably because of the wide distribution of dendritic cells and macrophages. CD209 positive cells were observed in human and cynomolgus liver and interpreted as Kupffer cells and/or portal macrophages. An unexpected staining of the central nervous system was observed and investigated using Western Blot. CD209 may be considered as an alternative to CD31 for confirmation of antigen preservation in human tissues. On the basis of these results we consider the performance of TCR studies in our laboratory to be fully validated.
67. Reproductive Toxicity In Male Rats By A Mixture Of Pesticides At “Low” Doses
Juliana Elaine Perobelli, Meire França Martinez, Carla Adriene Franchi, João Lauro Viana de Camargo, Wilma Kempinas
São Paulo State University (UNESP), Department of Pathology, TOXICAM, Botucatu, SP, Brazil
This study aimed to evaluate in rats the potential reproductive toxicity of a mixture of five pesticides: dicofol, dichlorvos, permethrin, endosulfan and dieldrin. These pesticides were chosen because their residues were found by the Brazilian Agency for Health Surveillance (ANVISA) in tomatoes regularly available over the counter to the Brazilian consumer. The pesticide levels in the diet were decided according to their toxicological critical doses to rats, as available in the open literature. Male Lewis rats (6 weeks old, 200g) were allocated to the following eight groups according to the diet: 1- Plain chow, negative control; 2 – “Low dose”, each pesticides mixed to the diet at their respective NOELs; 3 – “Effective dose”, each pesticides mixed to the diet at their respective LOAEL for dieldrin and endosulfan, LOEL for dicofol and LEL for dichlorvos and permethrin; groups 4a, 4b, 4c, 4d, 4e - positive controls, each subgroup received each pesticide individually, at their respective “effective” doses. After 8 weeks of feeding treatment the animals were sacrificed and reproductive parameters were evaluated. Sperm morphology, daily sperm production (DSP), sperm transit time through the epididymis, hormonal levels and histological evaluation of testis and epididymis did not present significant differences among the groups. On the other hand, sperm motility decreased (p<0.05) in animals of groups 2, 3, 4a and 4e when compared to negative control. These results suggest impairment of sperm quality and fertility potential in rats exposed to low doses of the studied pesticides, in spite of negative effects on DSP and spermatogenesis.
68. Characterization of Intestinal Ulceration, Impaired Spermiogenesis and Splenomegaly in the Delta-6 Desaturase (D6D) Null Mouse Due to Loss of Highly-Unsaturated Fatty Acids
Sandeep Akare1, Manabu Nakamura2,3, Manuel Roqueta-Rivera3, Chad Stroud2, Shir Gilor1, Mariangela Segre1, Rex Hess4, Wanda Haschek1
1Department of Pathobiology, University of Illinois at Urbana-Champaign, Urbana, Illinois, United States, 2Division of Nutritional Sciences, University of Illinois at Urbana-Champaign, Urbana, Illinois, United States, 3Department of Food Science & Human Nutrition, University of Illinois at Urbana-Champaign, Urbana, Illinois, United States, 4Department of Veterinary Biosciences, University of Illinois at Urbana-Champaign, Urbana, Illinois, United States
Delta-6 desaturase (D6D), encoded by the FADS2 gene, catalyzes the first step in the synthesis of highly-unsaturated fatty acids (HUFAs) such as arachidonic (AA), docosapentaenoic (DPA) and docosahexaenoic (DHA) acids, as well as the last desaturation of DPA and DHA. D6D-null mice (−/−) developed intestinal and skin ulceration, impaired spermiogenesis and splenomegaly at day 135. Splenomegaly was characterized by increased extramedullary hematopoiesis and myeloid hyperplasia and was accompanied by peripheral neutrophilia. In −/− males, normal spermatozoa and elongated spermatids were absent. Here we characterize the lesions in the intestinal and reproductive tract and investigate the cause of the splenomegaly. D6D-null (−/−) and +/+ male and female mice were fed a purified diet lacking D6D products, but containing ample linoleic acid (LA) to day 42. Male and female −/− mice developed erosion/ulceration of the intestinal mucosa, primarily at the ileocecal junction, while +/+ mice were unaffected. Skin ulceration and splenomegaly noted in the −/− mice at day 135 were not observed. Impaired spermiogenesis in the −/− males was characterized by markedly decreased numbers of normal spermatozoa and elongated spermatids in the seminiferous tubules and epididymis, while +/+ mice were unaffected. In conclusion, the splenomegaly was interpreted as a reactive change due to splenic extramedullary hematopoiesis and myeloid hyperplasia, secondary to dermal ulceration. In addition, AA but not DHA supplementation, prevented the intestinal erosions/ulcerations in −/− mice, suggesting a role of ischemia secondary to impaired prostaglandin synthesis (including PGE2) due to the lack of prostaglandin precursor, AA, in the −/− mice.
70. Quantitative Imaging Cytometry of Tissue Micro Arrays to Support Preclinical Drug Development
David Krull, Richard Peterson
GlaxoSmithKline, RTP, NC, United States
Preclinical drug development studies are conducted to evaluate various endpoints, such as efficacy, specificity and safety by examining protein expression and changes in tissue morphology. There is often interest in evaluating targeted proteins across multiple tissue types. A typical scenario would involve placing one or more tissues on a microscope slide then evaluating various immunohistochemistry (IHC) protocols. Approximately 10 to 15 slides would be needed to screen all tissue types of interest. Tissue Micro Arrays (TMA) containing normal or tumor samples from animal and human donors are an efficient tool to look at selected markers across multiple tissues using one slide. We have used whole mouse, rat and major human organ arrays. Here we demonstrate the use of dual IHC protocols on human multi-tumor arrays. Selected markers include CD31/CD34, cytokeratin 7/CD31, S100/GFAP, kappa/lambda (in situ hybridization) and Ki67/CD68. Protein expression in these arrays is measured using the iCyte® Automated Imaging Cytometer (Compucyte Corp., Westwood, MA) which utilizes lasers, PMTs and light absorption to quantify and evaluate antibody labeling (developed with chromatic and fluorescent dyes) across multiple tissue types in the TMAs. The instrument automatically identifies core elements, performs spectral deconvolution to isolate markers of interest, and quantifies the amount of expression of the markers. High resolution images of the cores are obtained simultaneously with quantitative analytical data. The development of protocols using TMAs and iCyte® quantification provide a useful standard for confirming antibody specificity in human TMAs with cross reactivity and specificity in rodent models.
71. Estimation of Intra-abdominal Adipose Tissue Volume in Mice Using Single Image Computerized Tomography
Brent Walling1,4, Luhua Zhang2, Aleksandra Michalowski1, Dan Schimel3, Jeeva Munasinghe3, R. Mark Simpson1
1Laboratory of Cancer Biology and Genetics, Center for Cancer Research, National Cancer Institute, Bethesda, MD, United States, 2Cell and Cancer Biology Branch, Center for Cancer Research, National Cancer Institute, Bethesda, MD, United States, 3Nuclear Magnetic Resonance Research Center, National Institute of Neurological Disorders and Stroke, Bethesda, MD, United States, 4Department of Pathobiology, University of Illinois, Urbana, IL, United States
Intra-abdominal adipose tissue (IAT) burden has been implicated as a risk factor for diabetes, cardiovascular disease, and certain cancers. IAT fat area derived from a single axial (transverse) view-plane computerized tomography (CT) image associated with a fixed anatomical abdominal landmark has been used in people for estimating IAT, increasing throughput while reducing radiation exposure. This study attempts to establish the suitability for estimating IAT from a single slice image in mice. Computer assisted image segmentation of IAT was performed on three strains of mice representing obese (B6.V-Lepob/J (Lepob)), trim (FVB), and lean (Azip) body fat conformations. IAT area from axial images was acquired at 3 mm intervals, starting at the 4th and 5th lumbar intervertebral space (L4-L5) as initial landmark. Total IAT volume was determined by interpolation between individual axial images using Amira software v5.2. Eleven area measurements from obese Lepob mice indicated mean area of IAT was greatest at L4-L5, however it predicted IAT volume poorly in contrast to studies in people. The axial image 12mm caudal to L4-L5 proved to be the optimal correlation between IAT area and volume measurements for obese mice, although this was not considered significant (R2 = 0.55, p = 0.153). Preliminary results imply use of axial slice area image analysis may not be a valuable means to predict IAT fat volume in obese mice. The approach may be useful for more physiological relevant IAT burdens. Analyses are ongoing for the mouse genotypes with typical or reduced amounts of IAT burden for comparisons.
72. Radiation Toxins And Mechanisms Of Radiation Toxicity In Development Of Radiation Sickness At High Dose, Dose-Rate, Low And High Linear Energy Transfer Radiation and Low Linear Energy Transfer Radiation Exposure
Vecheslav Maliev1, Dmitrij Popov1,2, Ludmila Malieva1, Viktor Zlobin1,4
1Russian Academy of Science, Vladicaucas, Russian Federation, 2Advanced Medical Technology and Systems Inc., Toronto, Canada, 3Russian Academy of Science, Vladicaucas, Russian Federation, 4Russian Academy of Informatics, Sankt-Petersburg, Russian Federation
Introduction: Radiation Toxins of irradiated mammals are a major cause of Multiple-organ failure (MOF) and Multiple-organ involvement (MOI), major inducing factor of inflammation and cytotoxic reactions. Radiation
Toxins (Specific Radiation Determinant – SRD) possess high toxic properties and are classified as Hematotoxic and Neurotoxic Radiation Toxins. Radiation Toxins play important role in development of Hematopoietic, Cerebrovascular, Cardiovascular and Gastro-Intestinal forms of Acute Radiation Syndromes. Four groups of Radiation Toxins were isolated from Central Lymphatic System of irradiated animals and have been designated as Neurotoxic Radiation Toxins (SRD-1) generated at Cerebrovascular ARS, Neurotoxic Radiation Toxins (SRD-2) generated at Cardiovascular ARS, Neurotoxic Radiation Toxins (SRD-3) generated at Gastro-Intestinal ARS, Hematotoxic Radiation Toxins (SRD-4) generated at Hematopoietic form of ARS.
Methods: Animals in experiments were irradiated with Low Linear Energy Transfer Radiation – γ-irradiation (PUMA-device), doses up to 10Gy and High Linear Energy Transfer Radiation – Fe56 irradiation at doses 3650-3820 Rad.
Results: Radiation Toxins (SRD-group) injected to healthy non-irradiated
Animals had initiated development of acute failure of systemic blood and lymph circulation and cytotoxic reaction with cell apoptosis development. Blood and lymphatic microcirculation were significantly destroyed.
Discussion: Clinical picture of Acute Radiation Syndromes depend on type and doses of Radiation Toxins. Form of Radiation Toxins depend on doses, time and type of radiation.