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SOCIETY OF TOXICOLOGIC PATHOLOGY’S 26TH ANNUAL SYMPOSIUM POSTER PRESENTATIONSP1 A Critical Comparison of Murine Pathology and Epidemiological Data of Dioxins and PCBs
1 Laboratory of Experimental Pathology, National Institute of Environmental Health Sciences, Research Triangle Park, NC, United States 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD, or dioxin) and dioxin-like compounds (DLCs) induce numerous toxicities, including developmental, endocrine, immunological, and multi-organ carcinogenic, in animals and/or humans. The National Toxicology Program (NTP) has conducted 4 murine carcinogenesis bioassays of TCDD in Swiss-Webster mice, Osborne-Mendel rats, B6C3F1 mice, and female Harlan Sprague-Dawley rats. Two carcinogenesis studies of 3,3’,4,4’,5-pentachloro-biphenyl (PCB 126) or 2,3,4,7,8,-pentachlorodibenzofuran (PeCDF) in female Harlan Sprague-Dawley rats were also conducted, because humans are exposed daily to a combination of DLCs, primarily via ingestion of food. The Toxic Equivalency Factor was developed to evaluate health hazards caused by these mixtures. Herein we review the pathological effects reported in humans exposed to TCDD, PCB 126, and PeCDF and compare them to similar effects seen in NTP murine studies performed with the same compounds. In the NTP studies, TCDD, PCB126, and/or PeCDF induced neoplastic lesions (hepatocellular and/or cholangiolar, pancreatic, thyroidal, and pulmonary) and nonneoplastic alterations (hepatocellular and/or cholangiolar lesions in the liver, squamous hyperplasia in the oral cavity, cardiovascular damage, thyroid hypertrophy, and immunosuppression) similar to those reported in humans. While differences in specific pathologies were observed, clear consistency could be seen in the target organs affected (liver, oral cavity, cardiovascular system, immune system, thyroid, pancreas, and lung) in studies of both human and rodent toxicity and carcinogenicity.
P2 Basal Cell Proliferation in a Preneoplastic Lesion of the Dog ProstateFaculdade de Medicina Veterinária e Zootecnia, Universidade de São Paulo, São Paulo, SP, Brazil Dogs are the only animal other than man to spontaneously develop prostatic intraepithelial neoplasia (PIN) and prostate cancer. Stem cells among the basal cell layer of the prostate are believed to play an important role in the failure of androgen-ablation therapy that occurs in the most advanced prostate cancer of man. The goal of this study was to investigate immunohistochemical markers to identify the cellular origin and the alterations of the pre-neoplastic lesions in the dog prostate. The prostate specimen was obtained at necropsy from a 7-year-old dog of mixed breed. Histologically, complex hyperplasia was seen. Foci of proliferation of epithelial cells lining preexisting ducts and acini were observed in cystic structures with crowding and heaping up of the epithelial cell layer. These lesions were first classified as PIN based on their morphological aspects. Immunohistochemistry was performed for high-molecular weight cytokeratin (HMC), PCNA, androgen receptor (AR), and for tyrosine kinase receptor c-erbB-2 antibodies. All foci of intraepithelial proliferations had marked expression of HMC and discrete nuclear AR staining. Moreover, PCNA-stained cells were more numerous in these lesions in relation to normal or hyperplastic acini. On the other hand, the proliferative lesions had a heterogeneous cytoplasmic c-erbB-2 expression ranging from negative to intense staining. The proliferative lesions shown in this study can be classified as basal cell hyperplasia with pre-neoplastic characteristics like cell pleomorphism, overexpression of PCNA, and heterogeneous expression of c-erbB-2. Androgen may play a role in the proliferation of basal cells seen in this case, as demonstrated by intense PCNA staining.
P3 Evaluation of the Carcinogenicity of Diisodecyl Phthalate (DIDP), a Plasticizer, in Rats
1 Toxicological Research Department, National Institute of Toxicological Research, KFDA, Seoul, South Korea and OECD and the American EPA have designated 2800 materials as High Production Volume Chemicals (HPV). These are defined as materials that are produced in excess of a million pounds in a single country. They also reported that there is little toxicological knowledge and few experiments with these materials. Diisodecyl phthalate (DIDP), a PVC plasticizer like other phthalate esters, is a major HPV chemical. DIDP, which has high physical flexibility, various colors, low viscosity, and high stability, is used as a coating material in balloons, vinyl ink, tents, textiles, homes, car interiors, and electric cable. These coating materials may contain up to 55% DIDP. In rodents, DIDP is suspected to be a peroxisome proliferator, a carcinogen. In this study, we evaluated toxicopathological risks of DIDP in a carcinogenicity test. F344 rats of both sexes (n = 416) were fed DIDP in diet at the levels of 0%, 0.04%, 0.2%, and 0.8% (w/w) for 104 weeks. Clinical signs, body weights, and food and water consumption were measured regularly. After treatment for 104 weeks, the animals were sacrificed and serological and histopathological examinations were performed. Survival at 104 weeks was markedly decreased in the high-dose group compared to the control group. Organ weights for liver, kidney, spleen, and brain were dose-dependently increased compared to the control group. RBC and WBC levels were increased for the low- and moderate-dose groups but decreased for the high-dose group. Histopathological examination indicated significantly increased incidences of mononuclear cell leukemia (MCL) at the high-dose compared to the control group. These results suggested that DIDP may play a role in the generation of MCL. However, since the F344 strain has a high incidence of spontaneous MCL, further research is necessary to evaluate the potential carcinogenicity of DIDP.
P4 Characterization of DNA Repair in Mice Congenic for the Polymorphic DNA Repair Gene Prkdc: Implications for Susceptibility to Breast Cancer
1 Colorado State University, Fort Collins, CO, United States and Investigations in our laboratory have described a heritable, naturally occurring susceptibility to radiogenic breast cancer in BALB/c mice. Significant phenotypic differences between BALB/c (susceptible) and C57BL/6 (resistant) strains have been linked to two unique single nucleotide polymorphisms (SNPs) in Prkdc-BALB. The SNPs encode two amino acid substitutions in the protein product, DNA-PKcs, which plays an integral role in bringing DNA ends together during V(D)J recombination and in non homologous end-joining, the primary DNA double-strand break (DSB) repair pathway. In studies using F1 hybrids (BALB x C57BL/6) and F1 x BALB backcross progeny, homozygosity for Prkdc-BALB was strongly correlated with susceptibility to radiation-induced chromosomal instability and ductal dysplasia.
We have employed a direct genetic approach to demonstrate a mechanistic linkage between the Prkdc locus and the BALB phenotype. We have developed and characterized two new inbred mouse strains that are congenic for Prkdc (B6.C-Prkdc: Prkdc-BALB allele on C57BL/6 background, and C.B6-Prkdc: Prkdc-B6 allele on BALB background) and are using these to determine the consequences of Prkdc-BALB expression on DNA-PKcs protein function in the context of radiation-induced DNA damage. The newly developed congenic strains have been characterized genetically for donor genome contamination by sequencing, DMit marker genotyping, and SNP microarray. Western analysis using congenic mice tissues has demonstrated that Prkdc-BALB decreases DNA-PKcs protein expression and impairs DNA DSB repair. Our results to date demonstrate that BALB/c polymorphisms in the DNA repair gene Prkdc are responsible for altered protein expression and DNA DSB repair, and support our hypothesis that Prkdc-BALB may initiate mammary carcinogenesis by inducing genomic instability. Study endpoints include total and specific kinase activity, in vivo DNA repair, and in vitro repair by disappearance of
P5 Columnar Cell Lesions in the Postmenopausal Primate BreastWake Forest University School of Medicine, Winston-Salem, NC, United States Columnar cell lesions (CCLs) are common morphologic alterations in the human breast epithelium, which may serve as precursors or markers for breast cancer. In this study, we characterized CCLs in a group of 63 aged postmenopausal monkeys (average age, 21.8 years) receiving one of three experimental diets for 8 months: (1) control casein/lactalbumin (C/L); (2) C/L with oral 17β-estradiol (E2) at a human dose equivalent of 1 mg/day; and (3) C/L with the soy isoflavone metabolite equol (EQ) at a human dose equivalent to 105 mg/day. The overall single-section prevalence of CCLs on mammary gland histology was 41% (26/63). Two distinct morphologic CCL subtypes were identified. Type 1 CCLs showed epithelial cell stratification, rudimentary lumen formation, and prominent myoepithelial cells, features similar to developing ducts. Type 2 CCLs had distinctive palisading densely packed nuclei, and cells often arranged into micropapillary structures. The prevalence of type 1 columnar cell hyperplasias was higher in animals treated with E2 (p = 0.01) but not EQ (p = 0.61), whereas type 2 hyperplasia prevalence was not affected by dietary treatment. Compared to normal ductal epithelium, type 1 but not type 2 CCL cells showed higher expression of estrogen receptor alpha (p < 0.01, all groups) and progesterone receptor (p < 0.01, E2 group only), and lower expression of the proliferation marker Ki67 (p < 0.01, E2 group only). These findings suggest that subtypes of CCLs may have different biologic behavior and potential importance as biomarkers of breast cancer.
P6 Comprehensive Expression Pattern of AP-1 Transcription Factor Family Proteins in the Serial Cellular Stages of Cholangiocarcinogenesis
1 School of Veterinary Medicine, Kangwon National University, Chuncheon, South Korea and Transcription factor AP-1 consisting of Fos and Jun proteins has strongly been suggested to be implicated in carcinogenesis by specifically regulating cell proliferation, differentiation, apoptosis, and oncogenic transformation. In the present study, we explored the expression pattern of each component of AP-1 at the serial cellular stages during cholangiocarcinogenesis in hamsters. We obtained precancerous lesions and biliary cancers from hamster cholangiocarcinoma (ChC) model and then performed immunohistochemical staining for c-Fos, c-Jun, Fra-1, Fra-2, and Fos B using ABC method, followed by semiquantitative analyses in the different stages of biliary cells during the carcinogenesis. As results, high frequency of strong nuclear expression of c-Fos was evident in dysplastic bile ducts (DBD) and ChCs, relatively high in hyperplastic bile ducts (HBD), and very low in normal bile ducts (NBD). Fra-1 was strongly expressed in DBD and ChCs as well as NBD and HBD. It was also of great interest that Fra-2 and Fos B, showing nuclear staining, were highly expressed in ChCs, which contrasted with DBD indicating low frequency of positive and negative NBD and HBD. On the other hand, nuclear immunoreactivity of c-Jun was increased in HBD, DBD, and ChCs. The results in some proteins were also supported by Western blot analyses. Our results strongly suggested that AP-1 family proteins are significantly and differentially implicated at any cellular stage during the developmental process of biliary cancer. This work was supported by the Korea Research Foundation Grant (KRF-2006-003-E00346).
P7 Diagnostic Criteria for Selected Peroxisome Proliferator-Activated Receptor (PPAR) Agonist-Induced Mesenchymal Lesions in the Rat
1 Experimental Pathology Laboratories, Inc., Research Triangle Park, NC, United States In 2005, the Health and Environmental Sciences Institute PPAR Agonist Project Committee was established to advance research on and to understand the modes of action and human relevance of this emerging rodent tumor data for PPAR agonists. A Pathology Working Group (PWG) was convened to develop consensus on morphologic criteria for tumor diagnoses and consistency of diagnoses across multiple studies for liposarcomas/ fibrosarcomas in rats. Participating institutions confidentially submitted multiple unstained slides from 99 rats for hematoxylin and eosin (H&E) staining and pathological review. Selected lesions were used for special histochemical and immunohistochemical staining. Lesions were most frequently submitted from subcutaneous and adipose tissues. Diagnoses involving proliferative lesions included fibrosarcoma, liposarcoma, malignant schwannoma, lipoma, hibernoma, fibroma, sarcoma, NOS, and hyperplasia, adipose tissue. Induced lesions often had myxomatous changes within the stroma that stained positively with Alcian blue. Distinction between PPAR-induced fibrosarcomas and liposarcomas was obscured in some cases by pleomorphic cell morphology, vacuolar changes of fibroblasts, a prominent fibrosarcomatous element within liposarcomas. For diagnosis of PPAR-induced lesions, final recommendations by the PWG consisted of slight modifications of the nomenclature and diagnostic criteria previously established by the STP. The routine use of histochemical and/or immunohistochemical stains was not recommended for the classification of these poorly differentiated sarcomatous neoplasms.
P8 Diagnostic Criteria for Selected Peroxisome Proliferator-Activated Receptor (PPAR) Agonist-Induced Vascular Lesions in the Mouse
1 Experimental Pathology Laboratories, Inc., Research Triangle Park, NC, United States In 2005, the Health and Environmental Sciences Institute (HESI) PPAR Agonist Project Committee was established to advance research on and to understand the modes of action and human relevance of this emerging rodent tumor data for PPAR agonists. A Pathology Working Group (PWG) was convened to develop consensus on morphologic criteria for tumor diagnoses and consistency of diagnoses across multiple studies for vascular changes and vascular proliferative lesions in mice and hamsters. Participating institutions confidentially submitted unstained slides from 420 mice and 10 hamsters for hematoxylin and eosin staining and pathological review. Tissues were most commonly submitted from the skin/subcutaneous tissue, liver, spleen, heart, and lymph node. Diagnoses involving proliferative lesions included hemangiosarcoma, hemangioma, angiolipoma, and angiomatous hyperplasia. Nonproliferative lesions included atrial thrombosis in the heart, congestion with hemorrhage in lymph nodes, and angiectasis, thrombosis, hemorrhage, and/or congestion in various organs. Lesions fairly unique to treatment with PPAR agonists included angiomatous hyperplasia and angiolipoma. In a few tumors that would have otherwise been classified as angiolipoma, regions of the vascular component appeared to be malignant, and were classified as hemangiosarcomas. With the exception of these two lesions, it was concluded by the PWG that the diagnostic criteria established by the STP for vascular neoplasms could be applied to PPAR-induced lesions, with slight modifications in nomenclature and criteria.
P9 Dietary Energy Restriction and Promotion of Liver Carcinogenesis by Hexachlorobenzene (HCB) in a Medium-Term Bioassay with Wistar RatsUNESP, Botucatu, Brazil The interaction between dietary energy restriction (DER) and exposure to the fungicide hexachlorobenzene (HCB) was studied in a rat liver carcinogenesis protocol that uses the development of putative preneoplastic glutathione S-transferase placental form positive (GST-P+) foci of hepatocytes as end-point. Male Wistar rats initiated with diethylnitrosamine (DEN; 200 mg/kg b.w.) were submitted to a 50% DER added or not with 50 ppm HCB during a 6-week period. A positive control group was fed HCB 150 ppm to document the promoting potential of the fungicide. Rats were sacrificed at the 8th and 52nd weeks. At the 8th week, DER-fed groups did not present expressive development of GST-P+ foci, irrespective the presence of HCB 50 ppm in the diet. Immunohistochemically evaluated apoptotic indices (cleaved caspase-3) were elevated in the liver of DER groups. On the contrary, the DEN+HCB 150 ppm group induced significantly more GST-P+ foci. At the 52nd week, combined incidence of hepatocellular adenomas and carcinomas was increased in this latter group. At the 8th week, rats fed DER+HCB 50 ppm presented the highest concentrations of HCB in the liver and the lowest levels in the adipose tissues; ad libitum–fed rats, treated with 50 or 150 ppm of HCB, showed the highest levels of HCB in peripheral fat depots. At the 52nd week, all HCB-treated groups presented higher fungicide levels in the fat tissues. These findings indicate that DER mobilizes HCB from the peripheral fat tissues but the transiently increased liver accumulation of HCB did not exert promoting influence on the hepatocarcinogenesis process.
P10 Effects of Consuming Claviceps Purpurea in Feedlot Cattle
1 EEA INTA Balcarce, Balcarce, Argentina The purpose of this study was to evaluate the effects produced by the ingestion of food rations with a concentration of 0.05%, 0.1%, and 0.5% of sclerotia of Claviceps purpurea. Twenty animals were chosen from a cattle herd and divided into 4 groups of 5 animals each. During a period of 50 days, the 4 groups were given different doses of sclerotia. These sclerotia contained 380 mg of ergoalkaloids stated as ergotamine/kg of sclerotia. Daily assessment of food intake, weight variations, respiratory frequency, body temperature, and dorsal skin thickness were made. Signs of the dysthermic syndrome were observed starting on the 8th day after initiation of the test and these included increased respiratory frequency, decreased food intake, increase in the search for water, hard breathing, and gasping. The group consuming the highest amount of sclerotia experienced a weight gain of 0.740 kg per day, whereas the control group gained 1.435 kg per day. The consumption of contaminated rations with Claviceps purpurea produces less weight gain in feedlot cattle, up to 50%.
P11 Evaluation of the Carcinogenic Mode of Action of Diuron [3-(3,4-Dichlorophenyl)-1, 1-Dimethylurea] on the Urinary Bladder of Male Wistar Rats1 UNESP Medical School, Botucatu, São Paulo, Brazil The herbicide Diuron [3-(3,4-dichlorophenyl)-1,1-dimethylurea], a substituted urea herbicide, is widely used in agricultural crops such as soy, cotton, and sugar cane. This herbicide was shown to exert carcinogenic activity on the urinary bladder mucosa of male rats when provided in high dietary concentration. Since the genotoxic and mutagenic potentials of Diuron are considered negative, its carcinogenic mode of action (MOA) was suggested to be non-genotoxic. Previous studies from our laboratory suggested that the MOA of Diuron on the urinary bladder mucosa of rats includes urothelial cell necrosis induced by urinary precipitates, crystals, and calculi, followed by regenerative enhanced cell proliferation and sustained urothelial hyperplasia that may favor neoplasia development. The present study aimed to evaluate the effects of reduced urinary pH on this potentially carcinogenic MOA. Thirty male Wistar rats six weeks old were allocated to three groups and received the following diets ad libitum: pelleted Nuvilab diet mixed with Diuron at 2500 ppm, Diuron 2500 ppm plus 10.000 NH4Cl, and the basal diet only during 15 weeks. The incidence of simple urothelial hyperplasia was significantly reduced in the 2500 ppm + 10.000 NH4Cl group (4/10 p < 0.014) when compared to the Diuron 2500 ppm group (8/10; p < 0.001). The present results indicate that urinary pH may protect the urinary bladder mucosa from the carcinogenic activity of high dietary dose of Diuron.
P12 Existence of Thresholds for In Vivo Mutagenicity and Carcinogenicity of Potassium Bromate in the Kidney of Rat
1 Japan Bioassay Research Center, Japan Industrial Safety and Health Association, Hadano, Japan and The purpose of the present studies was to investigate the dose-response relationship for in vivo mutagenicity and carcinogenicity of potassium bromate (KBrO3), a genotoxic carcinogen in the kidney of rats. To investigate in vivo mutagenicity of KBrO3 in the kidney, male Big Blue LacI transgenic rats were treated with KBrO3 at concentrations of 0, 0.02, 0.2, 2, 8, 30, 125, and 500 ppm in the drinking water, respectively. After 16 weeks of treatment, 500 ppm KBrO3 significantly increased the total mutation frequency and frequency of GC to TA transversion of the lacI gene in the kidney compared to control group, but 125 ppm and lower doses of KBrO3 had no effects. To investigate promoting potency of KBrO3 on kidney carcinogenesis, male Wistar rats were given 0.05% N-ethyl-N-hydroxyethylnitrosamin (EHEN) for 2 weeks in the drinking water to initiate kidney carcinogenesis. Thereafter, they were treated with KBrO3 at concentrations of 0, 0.02, 0.2, 2, 8, 30, 125, and 500 ppm (250 ppm after 12 weeks treatment) in the drinking water, respectively, for 24 weeks. There was no significant difference in both the number of preneoplastic lesions and the number of tumors in the kidneys in the 125 ppm or below KrBO3 groups, whereas both preneoplastic lesions and tumor numbers were significantly increased in the highest dose group. These results demonstrated the existence of no-effect levels for in vivo mutagenicity and promoting effects of KBrO3 on kidney carcinogenesis, indicating that thresholds exist for both mutagenicity and carcinogenicity of KBrO3 in rats.
P13 Expression of Ki-67 in the Uterus During Various Stages of the Estrous Cycle
1 Pfizer Global Research & Development, Ann Arbor, MI, United States and Rats have an average estrous cycle of 4–5 days. There are four phases (proestrus, estrus, metesterus, diestrus) in the estrous cycle in rodents. Histologic staging of the rodent estrous cycle is challenging and requires expertise. Thus, utilizing additional parameters such as cellular proliferation of the various components of the uterine microanatomy may assist with this process. Having an alternative method by which a pathologist can correctly identify the stages of the rodent estrous cycle would be valuable to the assessment and interpretation of safety studies for new drug candidates. This study was performed to investigate the microanatomic location of the uterine proliferative activity by image analysis and immunohistochemistry using Ki-67, a well-established marker of proliferating cells. Each stage of the rodent estrous cycle exhibited a different pattern of cellular proliferation. During proestrus, the lowest degree of cellular proliferation occurred in the glandular epithelial cells and the highest occurred in the myometrial cells. In estrus, lower levels of cellular proliferation were seen in the luminal and glandular epithelial cells, whereas a higher rate of proliferation occurred in myometrial cells followed by the stromal cells. At the metestrus stage, the highest cellular proliferation occurred in stromal and myometrial cells, whereas lesser proliferation was observed in luminal and glandular epithelial cells. This work demonstrates that in the rodent uterus there are cyclic changes in cellular proliferation in specific microanatomic uterine locations, which can aid in the staging of the estrous cycle.
P14 Fra-1 is a Potential Biomarker for Differential Diagnosis of Biliary Cancers in the LiverSchool of Veterinary Medicine, Kangwon National University, Chuncheon, South Korea Fra-1 protein is a member of the complex of AP-1 transcription factor that has been implicated in regulating cell proliferation and differentiation. As a driver of carcinogenesis, Fra-1 has been shown to be overexpressed differentially depending on the cancer cell type. To evaluate the potential importance of Fra-1 in the cholangiocarcinogenesis, we performed immunohistochemical staining for Fra-1 on the paraffin-embedded liver tissues of serial cellular stages of cholangiocarcinogenesis in hamsters. In addition, the liver samples with typical hyperplastic bile ducts, which were prepared in the hamsters by ligature of common bile duct for 12 days, were immunostained for Fra-1. Fra-1 was highly expressed in the cytoplasm and cell membrane of biliary cell populations including normal bile ducts, hyperplastic and dysplastic bile ducts, and primary and transplanted biliary cancer cells, without so much difference in the staining intensity. The expression of Fra-1 was independent of size of bile duct and shapes. Even hyperplastic single biliary cells were clearly detectable, which showed strong immunoreactivity for Fra-1. Smooth muscle cells of arteries were positive but relatively weak in expression of Fra-1. On the contrary, other liver cell populations including normal and altered hepatocytes, sinusoidal cells, and inflammatory cells were negative under our experimental protocol. Based on the results, we propose that Fra-1 could be a novel biomarker for differential diagnosis of cholangiocarcinomas in the liver. This work was supported by the Korea Research Foundation Grant (KRF-2006-003-E00346).
P15 Genistein-Induced Cell Death in Uterine Leiomyoma (UtLM) Cells Is Not by ApoptosisNational Institute of Environmental Health Sciences, Research Triangle Park, NC, United States Uterine leiomyomas (fibroids) are benign tumors of smooth muscle origin and the primary cause of hysterectomy in reproductive-aged women. Although the etiology of these tumors remains unknown, the overall lifetime exposure of women to estrogen is a known risk factor. The phytoestrogen, genistein, is an isoflavone found in soy products and shares structural similarities to17β-estradiol. In previous studies, we found that low doses of genistein stimulate proliferation of UtLM cells, but high doses result in inhibition of cell proliferation and induce cell death. In this study, we were interested in determining if apoptosis plays a role in genistein-induced cell death in UtLM cells. Using a Hoechst stain, we found that a high dose (50 µg/ml) of genistein induced significant apoptotic bodies in normal uterine smooth muscle cells (UtSMC), but not in UtLM cells. Also, when bcl-2:bak and bcl-2:bax protein ratios were analyzed, we found that the bcl-2:bax ratio was increased by approximately 1.6 fold in UtLM cells. Morphologically, UtLM cells treated with a high dose of genistein were sparsely arranged, fewer in number, and were shrunken. Ultrastructurally, UtLM cells had mitochondrial alterations such as swelling and membrane disruption, in addition to swelling of rough ER. Thus, based on these findings, we concluded that the inhibitory effect of genistein on UtLM cells might be due, in part, to mitochondrial toxicity, but not to significant apoptosis.
P16 Histomorphologic and Immunohistochemical Evaluation of Granular Cell Tumors in B6C3F1 Mice
1 Experimental Pathology Laboratories, Inc., Research Triangle Park, NC, United States and Granular cell tumors are most often benign neoplasms of uncertain origin. Four uterine granular cell tumors in control and treated female B6C3F1 mice were identified in chronic studies from the National Toxicology Program. Two tumors occurred in untreated control animals and two in treated animals receiving unrelated compounds. Tissue sections were evaluated histologically and stained with hematoxylin and eosin, periodic acid-Schiff with diastase resistance (PAS-D), Masson’s trichrome, toluidine blue, phosphotungstic acid-hematoxylin, and stained immunohistochemically with a panel of antibodies to muscle (desmin, alpha smooth muscle actin), neural (S-100, neuron-specific enolase), epithelial (wide-spectrum cytokeratin), and macrophage (F4/80) markers. The main histomorphologic feature of tumor cells was abundant eosinophilic cytoplasmic granules that stained positive for PAS-D. The tumors varied in histologic appearance and were composed of sheets and nests of round to polygonal cells with distinct borders. Nuclei were hyperchromatic, pleomorphic, and centrally to eccentrically located and often contained single nucleoli. Multinucleated giant cells were observed in two cases. Tumors were pale pink and homogeneous with trichrome stain and negative with toluidine blue. Three tumors had positive to weakly positive immunoreactivity for desmin, and one was positive for alpha smooth muscle actin. Expression of S-100, wide-spectrum cytokeratin, and neuron-specific enolase was negative for all tumors. Ultrastructurally, prominent electron dense cytoplasmic granules were abundant and contained secondary lysosomes with heterogeneous lysosomal contents. The histologic and immunohistochemical characteristics of these uterine granular cell tumors were suggestive of a myogenic origin.
P17 Immunohistochemical Analysis of β-Catenin, Cyclin D1, PCNA, and Cox-2 in Large Intestinal Proliferative Lesions of the F344 Rat Demonstrate that Nuclear β-Catenin May Represent a Marker for Differentiating Complex Hyperplastic Lesions from Neoplastic LesionsNational Institute of Environmental Health Sciences, Research Triangle Park, NC, United States β-catenin protein has two distinct roles in the cell. It is the central component of the cadherin cell adhesion complex and acts as a mediator of the Wnt/β-catenin signal transduction pathway facilitating transcription of target genes that encode effectors for control and regulation of the cell cycle. In colorectal cancer, nuclear accumulation of β-catenin activates transcription of cell cycle genes such as cyclin D1, resulting in increases in cell proliferation, invasion, and inhibition of apoptosis, all consequences that contribute to the development and advancement of colorectal cancer. The present study used immunohistochemical analysis to determine the expression patterns of β-catenin and other growth regulatory proteins in large intestinal hyperplasias, adenomas, and carcinomas from F344/N rats in 2-year toxicology and carcinogenesis studies. Nuclear accumulation of β-catenin protein was associated with adenomas and carcinomas but was not seen in hyperplasias, suggesting that nuclear β-catenin protein accumulation may be a useful marker for differentiating complex hyperplastic lesions from neoplastic lesions.
P18 Induction of Uncommon Reproductive Tract Tumors Following Intrauterine Administration of Quinacrine in a Two-Year Carcinogenicity Study in Rats
1 Huntingdon Life Sciences, East Millstone, NJ, United States Intrauterine administration of quinacrine has been used as an effective, easy, and inexpensive method of female sterilization in developing countries for over 25 years, but comprehensive data on long-term adverse effects are lacking. Quinacrine is both mutagenic and clastogenic in vitro, raising concerns about its carcinogenic potential. In the study reported here, rats were given two intrauterine doses of quinacrine 21 days apart at approximately 2x, 28x, 50x, and 70x the normal human dose, were observed for two years, and then were euthanized and necropsied. Standard tissues, including multiple sections of uterus, were examined microscopically. The only tissues showing macroscopic and microscopic changes associated with quinacrine were in the female reproductive tract. Considering the uterus, cervix, and vagina together, there was a dose-related increased incidence of edema, necrosis, cystic degenerative change, and myometrial hypertrophy in rats given quinacrine at 28x the human dose. At the same dose levels in these tissues, there also was an increased incidence of uncommon tumors (tumors with less than 1% historical background incidence) that included yolk sac carcinoma, mixed mullerian tumor, and carcinosarcoma. While the incidence of each individual type of uncommon neoplasm was very low, collectively, they occurred more frequently in groups given quinacrine at 28x the human dose. In contrast, the incidence of benign stromal polyp (considered a common tumor in this strain of rat), was decreased in quinacrine-treated rats. The poster discusses the non-neoplastic changes and the unusual range of uncommon reproductive tract tumors in response to quinacrine.
P19 Inflammatory and Angiogenic Marker Correlation with Histological Grade in Canine Meningiomas
1 Department of Veterinary Biosciences, The Ohio State University, Columbus, OH, United States Recent studies have implicated inflammatory mediators and microvascular proliferation as potential prognostic indicators in human meningiomas. Similar studies have not been reported in canine meningiomas, the most common primary brain tumor in that species. Additionally, no system has been described to grade canine meningiomas, a practice routinely performed for human tumors. This study describes a grading system for and reports the distribution of inflammatory and angiogenic markers in canine meningiomas. Surgical biopsies from 24 canine meningiomas were graded using a modification of the human World Health Organization system (grades I, II, and III) and assigned a histological subtype. Sections were immunohistochemically stained for markers of vascular proliferation (VEGF, Factor VIII related antigen), inflammation (Cox-2), and cellular proliferation (Ki67). Meningioma subtypes examined included meningothelial (11), psammomatous (4), transitional (2), mixed tumors (3), and others (fibroblastic, angioblastic, malignant meningothelial, microcystic). Psammomatous meningiomas were generally grade I, whereas meningothelial were split between grade I and II. VEGF staining intensity and distribution varied widely within given tumor types and grades, whereas microvascular density was higher in grade II tumors. Positive staining for Cox-2 was associated with grades II and III and localized to areas of inflammation, necrosis, and advancing edges of the tumor. Ki67 staining showed strong correlation with mitotic activity as assessed during tumor grading. This study demonstrates the distribution of Cox-2 and microvascular density in canine tumors and the correlation of those markers with higher tumor grades. These markers may prove useful as potential therapeutic targets and in predicting future clinical prognosis.
P20 N-methyl-N-nitrosourea: A Better Positive Control for p53+/-Mouse Carcinogenicity Studies
1 Pfizer, Groton, CT, United States This study evaluated the carcinogenic effects of N-methyl-N-nitrosourea (MNU) in B6.129-Trp53 tmlBrdN5 heterozygous (p53+/–) mice. Following one intraperitoneal injection of a 1% solution of MNU in citrate buffer (pH 4.5) at a dose of 75 mg/kg, 30 male and 30 female mice were observed for 6 months. Fifteen control mice of each sex were injected once with citrate buffer. Malignant lymphoma involving the thymus was observed in 24/30 males (80%) and 27/30 females (90%) treated with MNU. Malignant lymphoma of the spleen without thymic involvement was observed in one control female. Combined adenomas and carcinomas of the small intestine were found in 9/29 males and 5/29 females treated with MNU, but not in any control mice. Intestinal and gastric mucosal hyperplasia was seen more commonly in mice treated with MNU than in control mice. MNU produced loss of retinal photoreceptors and outer nuclear and plexiform layers in 27/29 males and all females. Malignant lymphoma caused the death of most mice treated with MNU before the end of the 6-month observation period. The high incidence of malignant lymphoma (~85%) in mice treated with MNU and the low incidence of malignant lymphoma in control mice indicate that: 1) MNU would be a more consistent and reliable positive control agent than p-cresidine for 6-month p53+/– mouse carcinogenicity studies; 2) 10 mice per sex treated with MNU would be an adequate group size to demonstrate a positive response; and 3) microscopic examination of mice treated with MNU could be limited to the thymus.
P21 OSU-HDAC42, a Novel Histone Deacetylase Inhibitor, Suppresses Prostate Tumor Growth and Causes Reversible Gonadal Degeneration In VivoThe Ohio State University, Columbus, OH, United States In light of the established link of aberrant epigenetic states with neoplasia, the targeting of histone deacetylases (HDAC) by small molecules is an active focus in the field of anticancer drug discovery. Here we evaluate the prostate chemopreventive and whole-body effects of dietary administration of OSU-HDAC42, a phenylbutyrate-based HDAC inhibitor synthesized in our laboratory. Tumor volumes from PC3 xenograft-bearing nude mice were compared between those treated with 208 ppm OSU-HDAC42 in the diet and those gavaged with a similar dose in a pilot study that preceded evaluation in the transgenic adenocarcinoma of the mouse prostate (TRAMP) model. At 6 weeks of age, TRAMP mice received AIN-76A diet with or without 208 ppm OSU-HDAC42 for 4 or 18 weeks and were analyzed with respect to prostate intraepithelial neoplasia (PIN) and carcinoma development, respectively. Systemic effects of drug treatment were determined by hematologic and histopathologic evaluation. The reversibility of observed lesions was characterized in age-matched wild-type animals. Diet and gavage-administered OSU-HDAC42, equivalently suppressed xenograft tumor volumes by 65.6% and 65.3% (p < 0.002) compared to controls, respectively. In TRAMP, despite equivocal differences in PIN development, the incidence of carcinoma was decreased from 100% (controls) to 10% following 18 weeks of drug treatment. Western analysis revealed potent target modulation in the prostate. Reversible gonadal degeneration characterized by depletion of germ cells occurred in drug-treated mice including females. OSU-HDAC42 has chemopreventive relevance in the progression of PIN to carcinoma in TRAMP and causes reversible gonadal degeneration by a currently undefined mechanism.
P22 Pfaffia Paniculata Roots Induce Apoptosis in Liver in Mouse Model of Hepatocarcinogenesis
1 Department of Pathology, Faculty of Veterinary Medicine and Zootechny, University of São Paulo, São Paulo, SP, Cidade Universitária, Brazil The Pfaffia paniculata root (PpR), Brazilian ginseng, has been used as tonic, aphrodisiac, and antidiabetic. Some studies have demonstrated its anticarcinogenic activities. We observed that PpR has an effect on hepatocarcinogenesis by reducing the incidence, size, and number of preneoplastic lesions. The aim of this research was to study the effects of PpP in mice liver cells in the carcinogenesis initiation step. We performed an experiment in which forty BALB/c male mice fifteen days old were separated in 4 groups. Three of these groups received diethylnitrosamine (DEN, 10 µg/g i.p.) and were treated for 27 weeks with PpR added to commercial food in different concentrations (0%, 2%, or 10%). The control group (CT) received only saline at day 15 and commercial food without PpR. At 27 weeks they were euthanized, the livers were weighed, and metacarn-fixed liver samples were processed. The apoptotic bodies per cm2 of liver were counted in H&E slides, under fluorescent microscopy. The groups DEN+0% or DEN± 10% showed a bigger body weight (18.3g ± 1.6; 16.5g ± 2.05, respectively) and liver relative weight (5.18g ± 10.4; 5.49g ± 0.90) in comparison to CT (13.01 g ± 3.72; 4.5 g ± 0.27). The DEN+10% group showed the highest apoptosis number (359.36 ± 144.83) in comparison to CT (16.71 ± 5.04) or DEN+0% (24.75 ± 12.76). These effects were dependent on fed PpR concentration. Our results showed that chronic treatment with PpR during the hepatocarcinogenesis initiation increased the cell death through apoptosis and can control the growth of liver tumors by this process.
P23 Review of NTP Studies for Mesenchymal Tumors and Nephroblastomas in the Rat Kidney
1 Experimental Pathology Laboratories, Inc., Research Triangle Park, NC, United States and Surveys from large databases of renal neoplasms of mesenchymal tissue origin and nephroblastomas, in the rat, appear to be few. The National Institute of Environmental Health Sciences/National Toxicology Program (NIEHS/NTP) conducts toxicological studies of chemicals of public health concern. The NTP database of 2-year bioassay and subchronic studies and the NTP Archives, containing specimens from those studies, represents a unique opportunity to review uncommon to rare neoplasms. Information was collected from approximately 207 2-year and 249 subchronic studies involving over 128,000 control and treated animals. The search was conducted primarily for Lipoma/ Liposarcoma; Renal Mesenchymal Tumor; Nephroblastoma; and others (Fibrosarcoma, Sarcoma NOS, Hemangioma/ Hemangiosarcoma, etc). Cases were reviewed to obtain information on sex, age, biological behavior, and, in many cases, to confirm the neoplasm histologically. The histopathologic diagnosis of these neoplasms is often problematic because: 1) many have similar histopathologic features, 2) malignant neoplasms often appear anaplastic, and 3) many contain entrapped but altered preexisting renal tubules. Although some neoplasms may grow to a large size, the metastatic potential appears low. Although renal mesenchymal tumor and nephroblastoma have been induced in the rat kidney by several carcinogens, apparently no neoplasm of mesenchymal tissue origin or nephroblastoma has been conclusively induced in a NTP bioassay study.
P24 STP Working Group for Historical Control Data of Proliferative Rodent Lesions
1 GlaxoSmithKline, King of Prussia, PA, United States The Historical Control Data Working Group, under the direction of the Scientific and Regulatory Policy Committee, is tasked with the preparation of a document that reviews current scientific practices, regulations, and relevant literature in order to provide best-practice recommendations for locating, generating, and applying microscopic historical control data to current long-term and carcinogenicity studies. The working group is focused specifically on historical control data of neoplastic and proliferative lesions, used in the safety assessment of nonclinical rodent chronic toxicity and carcinogenicity studies. The group is currently in the process of identifying, compiling, and evaluating available sources of historical control data. A survey was undertaken in the international toxicologic pathology community including representation across pharmaceutical, chemical, and government regulatory institutions to assess current practices within the scientific community, and the preliminary results are presented in this poster. The factors and variables that determine the reliability and usefulness of these data, relevant regulatory guidance, and selected literature will be reviewed and published in Toxicologic Pathology.
P25 The Polycomb Group Protein Bmi-1 Collaborates with H-Ras to Promote Transformation of Mammary Epithelial Cells and Development of Poorly Differentiated Metastatic Mammary Tumors In Vivo
1 National Cancer Institute, Bethesda, MD, United States and The polycomb group protein Bmi-1 is a transcription repressor reported to regulate self-renewal of normal and cancer stem cells and prevent cellular senescence through inhibition of cyclin-dependent kinase inhibitors p16Ink4a and p19Arf. Originally discovered as a cooperating oncogene with c-Myc in a murine model of leukemia, overexpression of Bmi-1 has since been reported in several forms of cancer, including breast cancer. A direct or collaborative role of Bmi-1 in the pathogenesis of breast cancer would be critical in the development of novel biomarkers for diagnosis and treatment of this disease. Overexpression of Bmi-1 alone and in combination with H-Ras in normal MEC (MCF10A) markedly changes cellular morphology, increases proliferative indices, and decreases the apoptotic response to apoptosis-inducing agents in vitro. Through the use of injection experiments in severe combined immunodeficient (SCID) and nude athymic mice, we show that Bmi-1 overexpression alone cannot induce mammary tumors, H-ras overexpression induces development of tumors with vascular, smooth muscle, and mast cell components, and the combination of Bmi-1 and H-Ras induces development of poorly differentiated aggressive mammary neoplasms that progress to spontaneous distant metastasis to liver, spleen, and brain. These findings suggest collaboration between H-Ras and Bmi-1 in the development of poorly differentiated metastatic mammary neoplasms with morphologic characteristics of epithelial-mesenchymal transition (EMT).
P26 Tubulin Binders Vinblastine, Vincristine, and Colchicine Cause an Interstitial Cell Cycle Arrest in the Rat MyocardiumHoffmann-La Roche, Nutley, NJ, United States The mechanism of cardiotoxicity of tubulin binders (TBs) has not been elucidated. We observed that a single administration of a TB in the rat caused an increase in the number of mitotic figures in the myocardial interstitium after 24 hours. We therefore hypothesized that interstitial cells are the primary target of TBs. To test this hypothesis, we evaluated the acute effects of a single intravenous administration of 3 reference TBs, vinblastine (0.5 and 3 mg/kg), vincristine (0.1 and 1 mg/kg), and colchicine (0.2 and 2 mg/kg). Mitotic arrest was identified at 24 hours in all 3 TBs high-dose groups based on an increase in the number of mitotic figures in the interstitium coupled with a dramatic decrease in the number of Ki67-positive interstitial cells. Analysis of the myocardial transcriptomic data further supported cell cycle arrest by identifying overrepresentation of the "G2/M DNA Damage Checkpoint Regulation" pathway at 6 h in the high-dose groups of all 3 compounds. Apoptotic figures and an increase in the number of cleaved caspase 3-positive cells were identified at 6 and 24 hours at the highest dose of each compound almost exclusively in interstitial cells; a few cardiomyocytes were affected as well. Transcriptomic data further suggested that some of the affected interstitial cells were endothelial cells based on the dysregulation of genes typically associated with vascular damage. Taken together, these data identify interstitial cells of the myocardium as an important target of the cardiotoxicity of TBs and identify cell cycle arrest as the mechanism of this toxicity.
P27 Virus-Associated Fibroproliferative Disorders in Cynomolgus Monkeys: A Histopathologic and Immunohistochemical Comparison with Human Kaposi’s Sarcoma
1 Schering-Plough Research Institute, Lafayette, NJ, United States and Retroperitoneal fibromatosis (RF) is an uncommon fibroproliferative disorder of non-human primates that shares epidemiologic and histopathologic similarities to human Kaposi Sarcoma (KS). As with KS in AIDS patients, both visceral RF and its subcutaneous variant are associated with immunodeficiency syndromes in monkeys infected with immunosuppressive viruses. AIDS-associated KS has been shown to result from co-infection with HIV and a recently described human gamma-herpesvirus (HHV-8). This poster compares the histopathologic and immunohistochemical findings of selected cases of human KS and simian visceral RF or subcutaneous fibromatosis. Formalin-fixed, paraffin-embedded samples of human and simian neoplasms were processed and stained with H&E, PAS, and Masson’s trichrome. Immunohistochemical stains for smooth muscle actin, desmin, vimentin, and CD-31 were used to characterize cell type, and samples were stained with ORF-73, a marker of HHV-8 infection, and for selected growth hormones, including VEGF, FGF-1, and IGF-1R. Histopathologically, the lesions of both species were characterized by well-differentiated to pleomorphic spindle cells. However, simian tumors tended to be more fibrous and lacked the vascular differentiation of KS. Both species’ specimens were positive for HHV-8, and both stained strongly for CD-31, VEGF, and vimentin, supporting a primitive mesenchymal-endothelial cell origin. Both species’tumors were mainly negative for smooth muscle actin and desmin, and mainly positive for IGF-1R. FGF-1 was expressed rarely on pleomorphic spindle cells only in RF. These results demonstrate similarities in etiology and pathogenesis for simian RF and human KS, and suggest that RF in monkeys may be a suitable model for studying KS.
P28 Air Pollution Reduces Somatic Cells and Phase VII in Mice TestisUniversity of São Paulo, São Paulo, SP, Brazil Air pollution has been associated with decreased semen quality in epidemiological studies. This work aimed to verify the effect of chronic exposure to air pollution on spermatogenesis in mice. Male mice BALB/c (n = 20) were exposed to urban air pollution in an exposure chamber 24 hours per day, 7 days per week, during a 4-month period (group A). Corresponding controls were housed in a similar chamber with filters for particulate matter (PM2, 5), during the same period (group B). After completing 120 days of exposure, the animals were sacrificed for testis collection and fixation in Bouin’s solution and later in alcohol 70%. Histological sections of the testis were stained with hematoxylineosin for quantitative morphological analyses of normal and degenerated cellular types within and between the seminiferous tubules. The tubular sections in phase VII of spermatogenesis were identified and scored in slides stained with Periodic Acid Schiff. Differences were considered significant when p < 0.05. The animals exposed to air pollution showed a reduction of the number of tubular sections in phases VII of spermatogenesis (p = 0.003), as well as a reduction of Leydig and Sertoli cell populations (p = 0.05 and p = 0.004, respectively). The germ cell populations showed no difference between groups. Air pollution can affect testicular somatic cells. Both Leydig and Sertoli somatic cells are supporters of normal germ cell development. Reduction of these cells could have adverse consequences on the number of tubular portions progressing through phase VII of germ cell maturation.
P29 Arsenite-Induced Apoptosis Is Prevented by Antioxidants in Zebrafish Liver Cell LineSeoul National University, Seoul, South Korea This study evaluated oxidative stress–induced apoptosis as a possible mode of action for arsenite toxicity in zebrafish liver cell line (ZFL cells). The heat shock protein 70 (HSP70), a chaperone protein, appears to provide protection against oxidative stress and apoptosis. Using the MTT assay, we demonstrated that survival of ZFL cells treated with arsenite for 24 h decreased in a dose-dependent manner. The possible mechanisms that promote the cytotoxicity of arsenite were evaluated. Cell viability assays revealed that arsenite caused a dose-dependent increase in cell death, and pretreatment of the ZFL cells with antioxidants blunted these effects. Antioxidants such as N-acetyl-cysteine (NAC, 5 mM) and dithiothreitol (DTT, 80 µM) significantly prevented arsenite-induced death of ZFL cells. Nuclear staining was performed using 1 µg/ml Hoechst, and cells were analyzed with a fluorescent microscope. 30 µM arsenite induced massive apoptosis that was identified by morphology and condensation and fragmentation of the nuclei of the ZFL cells. Pretreatment with NAC or DTT before arsenite insult effectively protected the cells against oxidative stress-induced apoptosis from the arsenite. Using a transfected human hsp 70 promoter-enhanced green fluorescent protein (EGFP) reporter, pHhsp70-EGFP, the induction of HSP70 against oxidative stress-induced apoptosis by arsenite was observed. The induction of HSP70 by arsenite increased in a dose-dependent manner, and pretreatment of transfected ZFL cells with NAC or DTT before arsenite insult reduced EGFP expression. Taken together, our results provide evidence that stimulation of the heat shock response is a sensitive biomarker of arsenic exposure and that arsenite causes oxidative stress–induced apoptosis in ZFL cells.
P30 Characterization of Age-Related Changes in the Spleen and Thymus from Control Cynomologus Macaques Used in Toxicity Studies
1 Pfizer, Global Research & Development, Ann Arbor, MI, United States and Identification of drug-associated immunomodulation in non-human primates based on weights and ratios and microscopic pathology of lymphoid tissues can be a challenge because of their normal variability. We characterized the age-related variability in the weights and ratios and histology of the spleens and thymuses obtained from control monkeys. Sections of the spleen and thymus from a total of 94 male and female Cynomolgus macaques of various age groups (<3 yrs; 3–6 yrs; 7–15 yrs) were evaluated. Organ weights related to body and brain weights, histology, and B- and T-cell immunohistochemistry (IHC) were evaluated quantitatively and semi-quantitatively. The splenic weights and ratios did not differ between various age groups or genders. By histologic examination of splenic sections, no differences in the number of germinal centers, periarterial lymphoid sheaths, or primary lymphoid follicles were seen. By IHC, no differences in B- and T-cell densities were seen in the spleen. In the thymus, there was a trend for a decrease in thymic weights and ratios with an increase in age that was more evident in males than females. In males, the interlobular fat significantly increased and the degree of delineation of the cortex and medulla significantly decreased in thymuses, but no similar trend was evident in females. The thymus cortex to medulla ratio significantly decreased with increasing age in males, whereas a trend for a decrease in cortex to medulla ratio, although not statistically significant, was present in females. B- and T-cell density did not change across various age groups or between genders.
P31 Chronic Inhalation of Crack Cocaine Affects Spermatogenesis in MiceUniversity of São Paulo, São Paulo, SP, Brazil In the present study, the effects of chronic inhalation of crack cocaine on the spermatogenesis of pubertal and mature mice were investigated. Balb/c mice of two different ages, young and adult (n = 20), were exposed to the smoke of 5 g of crack cocaine in an inhalation chamber for 5 days a week during 2 months. Corresponding control animals (n = 10) were kept in animal house during experimentation. Morphological quantitative analyses of testis were made in optical microscope. The spermatogenesis was evaluated during phase VII of maturation of the seminiferous epithelium. The number of spermatogenesis cell types (germ cells and Sertoli cells), the germ cell degenerations, and the intertubular Leydig cells population were scored. Differences were considered significant when p < 0.05. The number of tubular phase VII per testis showed significant (p = 0.023) reduction in young exposed animals. Significant reductions (p = 0.000) were observed in Sertoli cells and spermatids round and elongated (p = 0.001 and 0.005, respectively) in young intoxicated animals. Apoptosis is also increased in all intoxicated groups, being more severe in young groups (p = 0.000). Inhalation of crack cocaine smoke induced spermatogenesis disruption of chronic exposed mice. Crack toxicity was more severe in pubertal mice when sexual gonad undergoes maturation. We think that our findings should be of public health concern and that further investigations focusing similar effects on human males are warranted.
P32 Cisplatin Induces Renal Efflux Transporter Expression through Nrf2 Transcription Factor
1 University of Kansas Medical Center, Kansas City, KS, United States and The transcription factor NFE2-related factor 2 (Nrf2) mediates the inducible expression of cytoprotective genes, including NADPH: quinone oxidoreductase 1 (Nqo1), heme oxygenase-1 (Ho-1), and glutamate cysteine ligase (catalytic subunit, or Gclc) following electrophile exposure and oxidative stress. The use of the chemotherapeutic agent cisplatin is limited by nephrotoxicity. Administration of cisplatin to mice causes renal DNA adduct formation, generation of oxidative stress, and increased Ho-1 expression. Preliminary studies demonstrate that levels of the efflux transporters multi-drug resistance-associated protein transporters 2, 4, and 5 (Mrp2, Mrp4, Mrp5) and multi-drug resistance transporters (Mdr1a and 1b) are similarly higher in cisplatin-treated mice. It was hypothesized that induction of renal Mrp2, Mrp4, Mrp5, Mdr1a, and 1b expression following cisplatin is Nrf2-dependent. Plasma and kidneys from wild-type (WT) and Nrf2-null mice were collected 3 and 4 days after cisplatin (18 mg/kg i.p.). Nephrotoxicity (as determined by plasma BUN and cell proliferation immunohistochemistry) was greater in Nrf2-null compared to WT mice. As expected, induction of Nqo1, Ho-1, and Gclc mRNA and protein after cisplatin was dependent on Nrf2 expression. Similarly, cisplatin treatment increased renal Mrp2, Mrp4, Mrp5, and Mdr1b mRNA and protein in WT, but not Nrf2-null mice. Mrp1, Mrp3, and Mdr1a mRNA were not induced in either genotype. These results show that Nrf2 mediates renal induction of Mrp2, Mrp4, Mrp5, and Mdr1b after cisplatin. Coordinated regulation of detoxification enzymes and transporters by Nrf2 during cisplatin nephrotoxicity is a mechanism to remove potentially toxic mediators and promote proximal tubule cell recovery and proliferation.
P33 Classification and Frequency of Estrus Cycle in Beagles: A Retrospective StudyGlaxoSmithKline, Research Triangle Park, NC, United States The estrous cycle of the beagle is considerably longer with a luteal phase of approximately 75 days and an anestrus of 2–10 months. Theoretically, the in-life phase of a short-term (1–3 months) study can be completed within the metestrus or anestrus phase, and dogs in the same phase can be inadvertently assigned to one treatment group as a typical experiment group contains only 3 or 4 females. This can result in erroneous interpretation of drug effects on the cycle and, in turn, the organ weight data. This retrospective study was conducted to better understand the frequency of the different phases of the cycle in control beagles. H&E stained sections of ovary, uterus, cervix, vagina, and mammary gland were reviewed from 75 controls from 21 GLP studies, and the stage of the cycle was classified based on the morphology of the ovary and uterus. All studies were done at one facility (RTP). The average age for dogs reviewed in this study was 13.7 months, with a mean body weight of 6.91 kg. Based on histological classification, 42 dogs (56%) were in anestrus, 20 in metestrus (26.66%), 6 in proestrus (8%), 2 in estrus (2.66%), and 5 (6.66%) were classified as sexually immature. Mean ovarian weights were 0.795, 1.256, 0.963, 1.66, and 0.711 grams for dogs in anestrus, metestrus, proestrus, estrus, and immature dogs, respectively. The review indicates that more than 80% of the dogs can be in the anestrus-metestrus phase and stage of the cycle has a significant bearing on ovarian weight. In conclusion, interpretation of drug-induced effects on the estrus cycle and/or the morphologic changes in the reproductive tract should be performed with due consideration given to the stage of the cycle and the potential for a non-uniform assignment of dogs in different stages of estrus to drug treatment groups.
P34 Correlation of Drug-Induced Hepatocellular Hypertrophy with Cytochrome P450 Expression Using ImmunohistochemistryAllergan, Inc., Irvine, CA, United States Drug-induced hepatocellular hypertrophy may be subtle and difficult to detect by light microscopy. Specific proteins expressed during drug-related hepatocellular metabolic activities may serve as surrogate biomarkers of associated morphologic alterations. Here, we examine the relationship between drug-induced hepatocellular hypertrophy and the expression of cytochrome P450 (CYP) enzymes. Sprague Dawley (SD) rats were exposed to known CYP inducers: 3-methyl-cholanthrene (3-MC), phenobarbital (PB), pregnenolone-16-a-carbonitrile (PCN), isoniazid (ISN), and vehicle controls for four days. Liver morphology was evaluated by light microscopy, and correlation to CYP expression/induction (as determined by immunohistochemistry and microsomal assay) was examined. In comparison to vehicle controls, 3-MC, PB, PCN, and ISN induced minimal to mild centrilobular hepatocellular hypertrophy. This finding correlated well with immunohistochemistry (IHC) data showing that 3-MC induced mild expression of CYP1A (2.3-fold) or that PB induced moderate expression (3.7-fold) of CYP 2B and mild expression (2.3-fold) of CYP 3A. PCN and ISN caused only minimal expression (1.4- and 1.9-fold, respectively) of CYP2E. Microsomal assay data confirmed significant induction of CYP1A by 3-MC (24-fold) and CYP2B, 3A, and 2E by PB (9-, 2.2-, and 3.4-fold, respectively). These results indicate that drug-induced hepatocellular hypertrophy may be accompanied by correlating levels of CYP isozyme expression detectable by IHC. Thus, CYP IHC represents a potentially useful adjunct to H&E histopathology for comprehensive evaluation of drug-induced morphologic alterations in the liver.
P35 Cytotoxicity of Pyrrocidines in HepG2 Hepatocytes and PK15 Renal Cells
1 University of Illinois, Urbana, IL, United States and Pyrrocidines are newly reported polyketide-amino acid-derived antibiotics produced by Acremonium zeae, a prevalent seed-borne endophyte of corn. Pyrrocidines exhibit potent activity against Gram-positive bacteria and significant activity against Candida albicans, as well as fumonisin and alfatoxin producing fungi. Here we evaluate the effects of pyrrocidines A and B in two mammalian cell lines, HepG2 cells (derived from a human hepatocellular carcinoma) and PK15 cells (derived from a normal pig kidney). The H9C2(2-1) and HepG2 cells were incubated overnight to form monolayers and treated with 0 to 100 µM of pyrrocidine A and B for 24 hours. Cytotoxicity was evaluated by the MTT assay. Pyrrocidine A and B were cytotoxic to both HepG2 and PK15 cells after 24 hours of treatment. ED50 of pyrrocidine A to HepG2 cells was 0.66 ± 0.16 µg/mL and to PK15 cells was 1.03 ± 0.37 µg/mL. Pyrrocidine B was less potent than pyrrocidine A. ED50 of pyrrocidine B to HepG2 was 15.37 ± 4.47 µg/mL and to PK15 was 16.63 ± 5.47 µg/mL. The cytotoxicity of pyrrocidine A to HepG2 cells was more potent than other known mycotoxins, for example, deoxynivalenol ED50 = 8.36 µg/mL, fumonisin B1 ED50 > 100 µg/mL, zearalenone ED50 > 100 µg/mL, and moniliformin ED50 = 3.5 µg/mL. A sequential morphological study using time-lapse motion photography suggested that the cell death in PK15 cells induced by pyrrocidine A was consistent with apoptosis. This is the first report of toxicity in a mammalian system. In vivo studies are essential for risk assessment.
P36 Detailed Characterization of Cardiac Troponin Release and Histopathological Response Following Isoproterenol-Induced Myocardial Injury in the Rat
1 GlaxoSmithKline, Ware, Hertfordshire, United Kingdom This study aimed to provide a detailed time course characterization of cardiac troponin (cTn) release and histopathology responses associated with isoproterenol-induced acute myocardial injury in the rat. Male Hanover Wistar rats treated with a single subcutaneous injection of isoproterenol (0.1 mg/kg or 4 mg/kg) were necropsied (n = 5 per group) 0.5 to 72 hr later, along with concurrent controls. Blood was taken for serum cTn (I, T) analysis and hearts formalin-fixed/paraffin-embedded prior to sectioning. Serum cTn increased above concurrent control levels at 0.5 hr, peaked at 2 hr (0.1 mg/kg) to 3 hr (4 mg/kg), and declined to baseline by 48 hr (0.1 mg/kg) to 72 hr (4 mg/kg). Detailed characterization of histopathologic cardiac lesions utilized a variety of staining techniques (H&E, PTAH, MSB, cTnI immunohistochemistry), and a semi-quantitative severity score (minimal-marked) was based on the degree of observed myocardial damage. Myocardial degeneration was first observed at 0.5 hr post dosing (H&E). At 2–3 hr (maximal serum cTn levels) affected cardiomyocytes showed a loss of normal cTnI immunoreactivity, with pale areas of sarcoplasm and more intense staining in contraction bands. Lesions were dose-dependent in severity throughout the study, progressing temporally through myocardial degeneration/necrosis (6–24 hr), myocardial necrosis (48 hr) to myocardial fibrosis (72 hr), and reached maximum severity through the phase of declining cTn values. These results indicate that elevated serum cTn levels are an early indication of acute isoproterenol-induced myocardial injury and correlate with the development of early histologic lesions. Peak cTn levels then decline as histologic lesions progress from degeneration through necrosis to maximal severity.
P37 Doxil-Induced Cutaneous Lesions as a Model of Palmar-Plantar Erythrodysesthesia in Nude Mice
1 Medical Safety Research Laboratories, R&D Division and Palmar-Plantar Erythrodysesthesia (PPES) showing erythema, acanthosis, and cutaneous ulceration is a dose-limiting toxicity caused by infusion of certain anti-tumor drugs such as the pegylated liposomal doxorubicin (doxil). To establish a mouse model of PPES, doxil was intravenously administered at 10 mg/ kg/day, 3 times at a 4-day interval, or 4 times at a 7-day interval, to male BALB/cAnNCrj-nu/nu (nude) or its parent strain male BALB/c AnNCrj (hair) mice. The initial dosing day was designated as day 1 for the present study. As a result, decreases in body weight were observed in both strains from day 5 to 8. In nude mice, severe, erythema, erosion, and/or crust were observed in the interscapsular skin from day 14 on either regimen. Microscopically, the lesions were characterized as hyperkeratosis, acanthosis, ulceration with neutrophil and mast cell infiltration, atrophy of the follicle and sebaceous glands, and increased dermal collagen. The findings at the 7-day interval were somewhat more severe than those at the 4-day interval. In contrast, hairy mice displayed only unkept fur and milder lesions without mast cell infiltration. These results suggest that doxil-induced cutaneous lesions were more severe in nude mice than in hairy mice, and their morphological features resemble PPES in humans.
P38 Early Changes in Histopathology and Gene Expression in the Spleen of Rats with Drug-Induced Hemolytic Anemia
1 Astellas Pharma, Inc., Tokyo, Japan and It is important to detect the possibility of anemia—a serious adverse effect—in the early stages of drug development. When mild anemia is induced without bone marrow suppression, hemolytic anemia (HA) may be suspected; however, the pathological diagnosis is not easy since performing the clinicopathological examination at an optimal time is difficult. Various animal models of drug-induced HA were used to investigate the early changes in the histopathology and gene expression in the spleen. Acetylphenylhydrazine (APHZ; 10 mg/kg/day, subcutaneous) that induces unstable hemoglobin by forming Heinz bodies and ethylene glycol monobutyl ether (EGB; 100 mg/kg/day, per oral) that induces intravascular hemolysis by damaging the red blood cell (RBC) membranes were administered to induce mild anemia in F344 rats. Autopsies were performed at 6 and 24 h after a single dose. Immunohistochemistry was performed on the spleen to assess the expression of proteins such as heme oxygenase-1 (HO-1) and ferritin heavy chain (Fh); further, plasma haptoglobin was also evaluated. Gene expression in the spleen was analyzed using Affymetrix Rat Genome 230 2.0 Array. In APHZ-induced anemia, the expression of HO-1 and Fh progressed gradually due to Heinz body formation. In EGB-induced anemia, decreased plasma haptoglobin and increased expression of HO-1 and Fh due to RBC destruction were observed, which disappeared rapidly. The expression of the genes for monocyte activation and hematopoiesis was altered due to these chemicals. These changes in gene expression and immunohistopathology in the spleen might be useful for predicting drug-induced HA.
P39 Effects of Additive Estrogen Treatment and/or Ovariectomy on Age-Related Lesions in the Pancreatic Islet of Sprague-Dawley RatsDrug Safety Research Laboratory, Daiichi Sankyo Co. Ltd., Tokyo, Japan We have previously reported the increased occurrence of spontaneous hemorrhage in the pancreatic islet of male aging Sprague-Dawley (SD) rats as compared with females at identical ages. The hemorrhage appeared to be associated with decreased serum estrogen levels (Toxicol Pathol. 2007. in press). The present study was designed to elucidate the effect of additive estrogen treatment and/or ovariectomy (OVX) on the incidence of the islet lesions. Groups of 10 to 15 male and female SD rats were used; males were subcutaneously administrated β-estradiol 3-benzoate (EB) at 50 µg/animal once weekly for 20 weeks (7 to 26 weeks old), whereas females were subjected to OVX at 4 weeks old, and then given EB at 50 µg/animal for 20 weeks. These animals were sequentially sacrificed at 12, 18, and 26 weeks old, and serum estradiol level was measured in conjunction with pancreatic histopathology. At 26 weeks old, the islet hemorrhage in males receiving EB was significantly lower incidence than that in naïve males. Serum estradiol levels in these males were obviously higher than those in corresponding naïve males. In females with OVX, the hemorrhage was observed with high incidence from 12 weeks old, and additive treatment with EB recovered to the basal occurrence in the sham-operation. Increases in serum estradiol levels in the additive EB group were also observed. In conclusion, the onset of islet hemorrhage in SD rats was confirmed to largely depend on serum estrogen levels, which possessed the vascular protective action.
P40 Evidence of Urine Sediment and Urinalysis Differences between Wistar and Sprague-Dawley Rats Treated with Muraglitazar for 14 DaysGlaxoSmithKline, Research Triangle Park, NC, United States Urinalysis and sediment morphology/composition were evaluated following 14-day oral administration of Muraglitazar, a PPAR alpha/gamma agonist, at 50 and 100 mg/kg/day to male Harlan and Charles River Sprague-Dawley (HSD, CD-IGS) and Wistar rats. Urine was collected over 24 hours (0–4, 4–10, and 10–24 hours) on days 12–13 for urinalysis by automated chemistry analyzer. Sediment from free-catch urine collected in the AM on days 6 and 14 was evaluated by SEM with energy dispersive X-ray analysis. Sediment scoring evaluated: crystal abundance, crystal shape/length, and presence of mesh-like and/or granular material. Muraglitazar caused greater decreases in urine protein excretion and urine-specific gravity and greater increases in urine acidified total Ca in Wistar rats compared with Sprague-Dawley (SD) strains, and decreased urine citrate and increased urine pH regardless of strain. Mean sediment scores were increased over controls for HSD rats at both doses, CD IGS rats at 100 mg/kg/day, and there were no differences between treated and control Wistar rats. Elemental analysis of sediment showed crystals containing Mg, P, and K. SD rats had more abundant crystals that were increased in length, and associated with mesh-like material and/or granular material that contained Ca and P. Urine acidified total Ca in Wistar rats remained elevated over the sampling period consistent with less Ca precipitate formation. Increased urine protein excretion in SD rats combined with reduced citrate may permit Ca salts to form more microcrystalline precipitates. Differences in sediment and urinalysis between Wistar and SD rats treated with Muraglitazar might explain reduced urinary bladder tumorigenesis in Wistar rats treated with PPAR alpha/gamma agonists.
P41 Evidence that Caffeine Exacerbates Constitutive Androstane Receptor (CAR) Expression: Alterations in Body and Liver Weight, Cell Proliferation, Collagen, GAP Junctional Intercellular Communication, and CAR ExpressionLaboratory of Comparative and Experimental Oncology, Faculty of Veterinary Medicine and Animal Sciences, University of São Paulo, São Paulo, SP, Brazil Drug metabolism interactions are one of the greatest causes of hospital admissions, frequently leading to death. Caffeine-rich beverages (tea, coffee) are consumed worldwide, and it was shown that caffeine interacts with several drugs (e.g., acet-aminophen), increasing their side effects. The aim of this work is to provide evidence that caffeine increases the effects of the nuclear receptor CAR, induced by a specific ligand (TCPOBOP). Fifty animals were used and half received caffeine (50 mg/kg P.O.) daily for 18 days. On day 15, animals received TCPOBOP (6 mg/kg P.O.) and were euthanized at 0, 24, 48, and 72 hours. Body and liver weight were measured, and livers were metacarn-fixed, snap-frozen, or analyzed for GAP Junctional Intercellular Communication (GJIC). Histology, picrosirius analysis of collagen, PCNA immunohistochemistry, Western blot for PCNA, connexins (Cx) 26 and 32, and real time PCR for CAR were performed. We observed that caffeine decreased body weight after TCPOBOP time-dependently (p = 0.0026). In addition, caffeine increased liver weight after 48 h (p = 0.0004) and 72 h (p = 0.0002). Corroborating with these data, only 24 h after TCPOBOP, caffeine increased hepatocyte proliferation (p = 0.0350) and collagen expression (p = 0.0395). Also, a decrease in GJIC was noted after 24 h (p = 0.0084) and 72 h (p = 0.0335), and Cx26 and Cx32 expression correlated with the decrease in GJIC. Finally, caffeine increased 76% CAR expression after 24 h but not in other times. We concluded that caffeine clearly increases CAR expression and its effects in mice exposed to TCPOBOP. This information is important because CAR is a key regulator of metabolism of several drugs.
P42 Expression of Cannabinoid Receptor 1 in Skeletal and Cardiac Muscle
1 Bristol-Myers Squibb, New Brunswick, NJ, United States and The cannabinoid-1 receptor (CB1) is a G protein-coupled receptor that is highly expressed in the brain that mediates the orexigenic effects of endogenous cannabinoids. This has been borne out by studies showing that CB1 antagonists are efficacious anorexigenic agents. Additional studies have provided data for a metabolic effect of CB1 antagonists, with data supporting increased beta-oxidation of fatty acids. Since skeletal muscle is a major locus of fatty acid oxidation, we undertook a study to determine if CB1 is expressed in muscle tissue. Using Q-PCR and Western blotting, it was determined that expression of CB1 was very low or undetectable in skeletal and cardiac muscle from rat and dog, as well as in CB1-null (–/–) mouse brain. In contrast, high levels of CB1 expression were found in rat and dog cerebellum and wild-type mouse brain, tissues in which CB1-mediated pharmacological activity has been reported. Radioligand binding studies were also performed in membranes from skeletal and cardiac muscle tissue from dog. There was no detectable specific binding of cannabinoid radioligands to membrane preparations of skeletal muscle tissues (specifically semitendinosus, soleus, and diaphragm) or cardiac muscle tissues (atria and ventricle) from dog, whereas there were significant levels of radioligand binding observed in membranes from cerebellar tissue. Evaluation of rat and dog skeletal muscle and heart tissues by double-antibody immunofluorescence confirmed that CB1 was not expressed in these tissues, whereas there was considerable CB1 staining in rat and dog cerebellar Purkinje cells. Based on these analyses, the data indicate that the CB1 receptor is not expressed in detectable levels in skeletal or cardiac muscle from two species.
P43 Fumonisin Induced Pulmonary Edema in Swine: A Case Report
1 Department of Pathobiology, College of Veterinary Medicine, University of Illinois at Urbana-Champaign, Urbana, IL, United States and Fumonisins, mycotoxins produced by Fusarium verticillioides (F. moniliforme), frequently contaminate corn and can cause toxicosis in swine (porcine pulmonary edema, PPE) and horses (equine leukoencephalomalacia, ELEM). Fumonisins alter sphingolipid biosynthesis by inhibiting ceramide synthase. In swine, fumonisin-induced cardiovascular toxicity leads to pulmonary edema. Two 5-month cross-bred pigs from a group of 500 growers/finishers with 30% mortality were presented for necropsy following a recent change in feed. Findings consistent with severe pulmonary edema included pleural effusion and red, wet, and heavy lungs with markedly distended interlobular septa. On histopathology, alveolar spaces were filled with serous fluid, fibrin, and foamy macrophages. Alveolar septa were mildly thickened due to serous fluid and fibrin, and vasculature was congested. Interlobular septa and pleura were markedly edematous with fibrin on the pleural surface. Fumonisins B1 (FB1) and 5 ppm) and B2 (FB2) were detected in stomach contents (FB1 > feed (FB1 = 23 ppm; FB2 = 6 ppm). Serum analysis by HPLC showed markedly elevated sphinganine (average 5.30 µM; experimental control reference range 0.00–0.06 µM) and sphingosine (average 1.67 µM; experimental control reference range 0.01–0.07 µM) concentration as well as the sphinganine to sphingosine ratio (average 3.22; experimental control reference range 0.00–0.18) confirming fumonisin myocotoxicosis. Current U.S. FDA guidelines recommend a maximum total fumonisin level of 10 ppm in swine feed. The last PPE outbreak reported in the United States was in 1989. This report documents a recent PPE outbreak and demonstrates the utility of serum sphinganine and sphingosine levels in diagnosis.
P44 Guarana (Paullinia Cupana) Shows Hepatotoxicity in BALB/c but not in C57Bl/6 Mice: Effects on Liver Weight, Serum Alanine-Aminotransferase, and CholesterolFaculty of Veterinary Medicine and Animal Sciences, São Paulo, SP, Brazil Guarana is consumed worldwide as a tonic and stimulant, mostly because of high levels of caffeine (2.5%~5.0%) and polyphenols (13%~16%). Earlier, we reported a chemopreventive effect of guarana and protection against DEN-induced DNA damage. Also, guarana increases constitutive androstane receptor (CAR), and its effects are probably due to the caffeine content. In this study, we report that guarana could be hepatotoxic for certain types of mouse strains. We used BALB/c and C57Bl/6 female mice that were treated daily for 14 days with guarana (2000 mg/kg, P.O.). The mice were euthanatizied and liver and body weight data collected. Liver tissues were fixed in metacarn, and serum was collected to analyze ALT, cholesterol, and triglycerides. Histopathology was performed on H&E-stained slides. No differences in body weight and histopathology were noted between controls and treated mice. However, in BALB/c mice, liver weight (p = 0.0013) and ALT levels (p = 0.0141) were increased, indicating hepatotoxicity. However, no changes in serum cholesterol were noted (p = 0.2878). On the other hand, C57BL/6 mice showed no alterations in liver weight (p = 0.6278) and ALT levels (p = 0.5302) but showed decreased serum cholesterol (p = 0.0449). These observations suggest that these differential effects are probably due to differences in caffeine metabolism between strains, where BALB/c mice were found to be more sensitive to guarana hepatotoxic effects. Regarding cholesterol levels, it can be postulated that caffeine is probably less metabolized in C57BL/6 mice, allowing it to act on adenosine receptors and burning body fat. This work is fundamental because it shows that guarana could present differential effects depending on the mice strain used.
P45 Histomorphometric Characterization and Immunohistochemical Analysis of Steroid Hormone Receptor and Aromatase in Skin of the Göttingen Minipig
1 Michigan State University, East Lansing, MI, United States and
The Göttingen minipig is regarded as a useful animal model for investigative toxicity studies for many reasons, one of the most important of which is that this species is considered to share many anatomic and physiologic similarities with humans. Steroid hormones are known to regulate a number of important physiologic pathways in human skin. However, in the minipig, there is a lack of detailed descriptions of the histomorphometric features and detailed analysis of the actions of steroid hormones in the skin. This study evaluated skin characteristics with respect to variations in age, sex, and anatomic localization and characterized receptor distribution in minipigs using immunohistochemical stains for estrogen receptor (ER) –
P46 Histopathological Features of Capillaria hepatica Infection in Laboratory Rabbits
1 Huntingdon Life Sciences, Woolley Huntingdon, Cambridgeshire, United Kingdom and Capillaria hepatica is a nematode parasite of mammals with a worldwide distribution. The usual hosts for adult worms are rodents, but infections have also been reported in many other species. Although C. hepatica has been recorded in wild lagomorphs, no detailed histopathology has been described in wild, domestic, or laboratory rabbits. The outbreak reported here was confined to a group of 160 New Zealand white rabbits of both sexes, between 9 and 22 weeks of age, obtained from a commercial supplier. None of the animals exhibited adverse clinical signs. However, at necropsy, firm pale masses or pale areas were noted in the livers of a proportion of the animals. On histopathological examination, lesions were observed predominantly in the portal tracts. The lesions comprised dilated bile ducts (containing occasional adult worms and amorphous debris), peribiliary fibrosis, and lymphoplasmacytic infiltrates. Large granulomas, containing characteristic eggs and numerous macrophages, were also present in the portal areas. In the gall bladder, eggs were observed lying free within the lumen. The life cycle of C. hepatica is unique as it is thought to be the only nematode that inhabits the liver in the adult phase. Accordingly, it has been believed that entrapped eggs are generally liberated only after the ingestion and digestion of the infected liver by another animal. However, the presence of eggs in the biliary system in the present outbreak, but not described previously, indicates that a fecal-oral route of transmission for C. hepatica in the rabbit may be possible.
P47 Increased Connective Tissue Extracellular Matrix in the op/op Model of OsteopetrosisPfizer Global Research & Development, Ann Arbor, MI, United States Mice homozygous for the recessive osteopetrosis spontaneous mutation (op/op) have severe monocyte deficiency and develop severe osteopetrosis due to a defect in the production of macrophage colony-stimulating factor (M-CSF) and a deficiency in monocyte-derived osteoclasts. Here we describe a soft tissue finding in an osteopetrosis (B6C3Fe a/a-Csf1op/J) mouse model. Tissues were obtained at the time of necropsy from B6C3Fe a/a-Csf1op/J mice and age-matched wild-type mice, fixed in formalin, processed for hematoxylin and eosin sections, and light microscopic comprehensive tissue evaluation was performed. Mutant mice showed the classic traits of missing incisors and domed skull characteristic of op/op deficiency. Histologically, the bone marrow cavity was effaced by interweaving thick bony trabeculae consistent with osteopetrosis. An increase in a finely granular, basophilic interstitial extracellular matrix (ECM) was observed in the subcutaneous connective tissue of the op/op mice when compared with controls. Histochemically, the ECM was periodic acid Schiff (PAS) negative and stained dark blue with Alcian blue at a pH of 2.5, but not at 1 or 0.4 pH, indicating that it is composed mostly of non-sulfated glycosaminoglycans (GAGs). Our work suggests that in op/op mice there is increased ECM that is composed mainly of GAGs located in the subcutaneous tissue. This increase in the ECM may be related to altered matrix production or turnover because of changes in M-CSF production.
P48 Megakaryocytic Leukemia in a MouseHuntingdon Life Sciences, Huntingdon, Cambridgeshire, United Kingdom Neoplasia involving the platelet-forming element of the bone marrow has been recorded in humans, where the neoplastic elements have been identifiable as atypical megakaryocytes with myelogenous proliferation. Neoplastic lesions induced by recombinant retrovirus or whole body irradiation, developing from megakaryocytic cell lineage, have been recorded in mice. A single female CD-1 mouse killed on Day 657 of an oncogenicity study was diagnosed as having megakaryocytic leukemia. Abundant atypical (neoplastic) megakaryocytes were seen in the sinusoids of the liver, the spleen, bone marrow, and lymph nodes. Evidence of the neoplastic lesion was also seen in the adrenals, brain, gastrointestinal tract, heart, kidney, lung, mammary areas, ovaries, thymus, and uterus.
P49 Novel Vascular Lesions in Mice Given a Non-Peptide Vitronectin Receptor Antagonist
1 GlaxoSmithKline, King of Prussia, PA, United States and
Novel vascular lesions and secondary ischemic changes are described that were observed in mice treated with an integrin antagonist, SB-273005, a potent orally active nonpeptide vitronectin ( SB-273005 caused VSMC necrosis in aorta and renal arteries of mice. Lesions did not progress or recover, but there was medial hypertrophic adaptation even with continued dosing. This is considered direct species-specific VSMC toxicity of unknown mechanism and unrelated to vitronectin receptor antagonism.
P50 Procedural-Induced Acute Glandular Stomach Erosions in RatsJohnson & Johnson Pharmaceutical Research & Development, Raritan, NJ, United States Glandular stomach erosions can be induced by a variety of agents and stressful conditions, and synergy between aspirin and stress has been documented (Rainsford KD. Agents and Actions 5:553–558, 1975). Due to an incidence of acute glandular stomach erosions of about 10% in control female rats and about 2% in males in our facility (regardless of study duration) with an occasionally higher incidence in rats treated with test compounds, we investigated factors inducing these acute erosions. In the first study, 120 female rats were divided into 12 groups to identify potential external factors including handling stress, anesthetics, blood collection, and overnight fasting. Diclofenac was used as a positive control in this 4-day study. It was found that overnight fasting with blood collection (2.5 mL) was responsible for acute erosions, and (as expected) diclofenac increased the incidence of erosions (including ulcers), including erosions/ulcers appearing to be of several days duration based on the presence of fibroplasia. In a second study with 120 rats testing the effects of overnight fasting vs. blood collection under CO2 anesthesia, only the combination of overnight fasting with blood collection caused acute erosions. It was concluded that the erosions were occurring between the time of morning blood collection and necropsy later in the day, and that factors (e.g., stress) associated with various test compounds could potentially act synergistically with the overnight fasting and blood collection.
P51 Pulmonary and Cardio-Renal Inducible Nitric Oxide Synthase Expression in a Human Hypertension Model
1 Pfizer Global Research & Development, Ann Arbor, MI, United States and Hypertensive mice that express the human renin and angio-tensinogen genes are used as a model for human hypertension because they develop hypertension secondary to increased renin-angiotensin system activity. The role of inducible nitric oxide (iNOS) in this model is not understood. The goal of this study was to investigate, using immunohistochemistry, the cellular localization and distribution of iNOS in the lung, heart, and kidney from a mouse model of human hypertension using male and female double transgenic mice. The highest renal expression of iNOS occurred in cortical and medullary collecting ducts and distal convoluted tubules. No iNOS expression was present in vascular smooth muscle cells, endothelial cells, and glomeruli. iNOS expression ranged from minimal to mild in other renal micro-anatomic locations. In the lung, bronchial and vascular smooth muscle cells had moderate iNOS expression, with other pulmonary micro-anatomic locations either lacking or having minimal to mild iNOS expression. Marked iNOS expression was present in cardiac myocytes, whereas no expression was present in cardiac vascular smooth muscle cells or endothelial cells. iNOS expression was similar between males and females. Our work suggests that in hypertensive mice, (a) there are significant micro-anatomic variations in the pulmonary, renal, and cardiac distribution and cellular localization of iNOS, and (b) no differences in expression exist between genders.
P52 Pulmonary Lesions in Female Harlan SD Rats Following Two-Year Oral Treatment with Dioxins
1 Astellas Pharma, Inc., Osaka, Japan Dioxin and dioxin-related compounds have been associated with high incidences of pulmonary dysfunctions and/or cancers in humans. To evaluate the effects and relative potencies of these compounds, the National Toxicology Program completed a series of 2-year bioassays conducted on female Harlan Sprague-Dawley rats. They were treated orally for up to 2 years with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD); 3,3’,4,4’,5-pentachlorobiphenyl (PCB126); 2,3,4,7,8-pentachlorodibenzofuran (PeCDF); and a tertiary mixture of TCDD, PCB126, and PeCDF. In addition to treatment-related effects reported in other organs, a variety of pulmonary lesions related to exposure were observed. Pulmonary CYP1A1-associated 7-ethoxyresorufin-O-deethylase (EROD) activity was increased in all dosed groups. In all studies, the most common non-neoplastic lesions were bronchiolar metaplasia and squamous metaplasia of the alveolar epithelium, and the most commonly observed neoplasm was cystic keratinizing epithelioma. A low incidence of squamous cell carcinoma was associated only with PCB126 treatment. Potential mechanisms leading to altered differentiation and/or proliferation of bronchiolar and alveolar epithelia may function via CYP1A1 induction or disruption of retinoid metabolism.
P53 Reproductive Lesions in Female Harlan Sprague-Dawley Rats Following Two-Year Oral Treatment with Dioxin and Dioxin-Like Compounds
1 Astellas Pharma, Inc., Osaka, Japan Previously published animal studies suggest that prenatal and postnatal exposure to dioxins may profoundly affect the female reproductive system via endocrine disruption. We evaluated the reproductive toxicity and carcinogenicity induced by two-year exposure of female Harlan Sprague-Dawley rats by gavage to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD); 3,3’,4,4’,5-pentachlorobiphenyl (PCB126); 2,3,4,7,8-pentachlorodibenzofuran (PeCDF); 2,2’,4,4’,5,5’-hexachlorobiphenyl (PCB153); a tertiary mixture of TCDD, PCB126, and PeCDF; or a binary mixture of PCB126 and 153. Treatment-related changes included chronic active inflammation in the ovary that occurred in the PCB153 and binary-mixture studies of PCB126 and 153. Increases in the incidences of acute or chronic active inflammation of uterus were observed in all studies. The incidences of cystic endometrial hyperplasia were marginally increased in the PeCDF study. The incidences of squamous metaplasia in the uterus were significantly increased in the PeCDF and tertiary-mixture studies of TCDD, PCB126, and PeCDF. The incidences of uterine squamous cell carcinoma were significantly increased in the TCDD study. The incidences of uterine carcinoma were marginally increased in the PeCDF study. In the studies of PCB126, PCB153, the binary mixture, and the tertiary mixture, no increased incidence of any reproductive tumors was observed. The range of changes seen with the different compounds suggests that more than one mechanism may have been involved in promoting the female reproductive pathology.
P54 Sequential Changes in Oleic Acid-Induced Lung Injury in Beagles
1 Shin Nippon Biomedical Laboratories, Ltd. (SNBL), Kagoshima, Japan We previously reported on oleic acid (OA)-induced lung injury in cynomolgus monkeys (CM), a useful model of acute lung injury to study the effectiveness of a non-invasive imaging technique computed tomography (CT). In the present study, to clarify OA-induced lung injury development in dogs, we investigated sequential changes in blood gas, CT imaging and histology. Six beagles were injected intravenously with a single dose of OA (0.08 mL/kg) and sacrificed at 4h, 24h, Day 3, or Day 14 (n = 2, 1, 2, 1, respectively). CT images, blood gases, and histological changes were monitored periodically. Another 6 animals were injected with physiological saline as the control. OA at 0.08 mL/kg was non-lethal in beagles although mortality was observed in 2/9 CM. At 4h and 24h after OA injection, edema, hemorrhage, congestion, fibrin deposition, inflammation, and alveolar wall necrosis were observed in the lungs. Some lesions were spread in a wide area in the lung lobes, and were most severe in the subpleural area. At Day 3, marked proliferation of type II pneumocytes was observed. At Day 14, multi-focal fibrosis was observed in the subpleural alveolar or central alveolar area although the other lesions in the lung had mostly disappeared. Pao2 changes and CT findings corresponded to the results for CM. OA-induced lung injuries in beagles were developed in almost the same way as in CM. However, the lung lesions in beagles were more severe than those in CM, which was possibly caused by a physiological and/or functional difference. The effectiveness of the non-invasive, diagnostic method using CT imaging was confirmed with beagles.
P55 Spleen Damage in Response to Pteridium Aquilinum Administration in Mice
1 Faculty of Veterinary Medicine and Animal Sciences, Sao Paulo, SP, Brazil and Pteridium aquilinum (Pa), one of the most widespread plants on earth, is known to cause cancer in animals and humans when consumed. Our earlier studies have shown that Pa causes lymphoid organs injury, decreases bone marrow cellularity, reduces DTH response and NK cell cytotoxicity in mice. In this study, we evaluated spleen histopathology and the lymphocyte population in spleen. For histopathological evaluation, 9 C57BL/6 male mice were used per group and treated by gavage up to 14 days as follows: control (Co)0.0, Pa (P)30.0 gPa/Kg BW. Spleen was collected immediately after sacrifice and fixed in 10% formalin; tissue sections were stained with H&E. In another study, for the analysis of spleenic lymphocyte population, 4 C57BL/6 male mice were used per group and the same protocol was utilized for treatment. At sacrifice, the spleen was removed to prepare cell suspensions. From the cells obtained in each mouse, 1x106 cells/tube were incubated for 1h with either anti-CD4 or anti-CD8 or anti-CD19 conjugated with phycoerythrin. The samples were measured by flow-cytometry. Pa treatment reduced splenic lymphocytes (two-tailed p = 0.004) Co:0.32 (0.29–0.34) P:0.29 (0.22–0.32), Mann-Whitney test. However, no alteration in the different population lymphocytes was observed, which demonstrates that there was a reduction in all lymphocytes analyzed. These results indicate that Pa causes reduction of T and B lymphocytes in mice. Accordingly, reduced lymphocytes contribute to the carcinogenicity induced by this plant. Further experiments are in progress to study the immunomodulatory effects of Pa.
P56 Strain-Related Differences in Urine Composition of Male Rats of Potential Relevance to Urolithiasis
1 Bristol-Myers Squibb Research and Development, Evansville, IN, United States and
In rodent carcinogenicity studies with PPAR
P57 Subchronic Administration of Indole-3-Carbinol in B6C3F1 Mice and Fischer 344 Rats Is Associated with Increased Hepatic CYP 1A1 and 1A2 Activities, but without Liver Toxicity
1 National Institute of Environmental Health Sciences, Research Triangle Park, NC, United States Indole-3-carbinol (I3C), a component of cruciferous vegetables, can reduce the risk of certain cancers induced by several known carcinogens in models, but can also function as an initiator and tumor promoter. Similar to "dioxin-like" compounds, I3C induces hepatic and pulmonary cytochrome P450 1A enzymes through the Ah receptor, a process often associated with toxicity. In the current study, Fischer 344 rats were exposed to up to 300 mg I3C/kg body weight and B6C3F1 mice were exposed to up to 250 mg I3C/kg body weight five days a week for 13 weeks by gavage. No effects were observed on body weight, survival, clinical chemistry, hematology, or gross pathology. Dose-dependent increases were observed in liver weights in male and female rats and mice. In mice, hepatic CYP 1A1 and 1A2 activities were maximally increased 2- to 4-fold. In rats, hepatic CYP 1A1 activity was dose-dependently increased with a maximal induction of 109- and 81-fold in males and females, respectively, at 300 mg/kg. Hepatic CYP 1A2 activity was dose-dependently increased with a maximal induction of approximately 5-fold in both males and females. CYP 1A1 activity maximally induced by I3C in female rats exceeded previously observed activities induced by TCDD at doses that induced neoplastic and nonneoplastic lesions in multiple tissues at 2 years, including moderate to marked hepatotoxicity and liver tumors. The 2-year studies will provide further insight into the relationship between marked CYP 1A1 and 1A2 induction and liver tumor development in rats.
P58 Testicular and Epididymal Findings in Control Beagle Dogs
1 Pfizer, Groton, CT, United States Preclinical evaluation of chemically induced histopathologic lesions requires thorough knowledge of spontaneous and age-related spontaneous findings. The purpose of this study was to determine the incidence, type, and severity of spontaneous testicular and epididymal microscopic findings in 80 control beagle dogs (ages 6 to 36 months) from toxicology studies conducted between 2000 and 2005. Focal or diffuse testicular tubular hypospermatogenesis was observed in 87.5% of sexually immature dogs (6 to 8 months) and 21.7%–40% of sexually mature dogs (8 months to 3 years). The "germ cell depletion" pattern of hypospermatogenesis was characterized by variable and segmental decreases in numbers of spermatids and/or spermatocytes within seminiferous tubules. A second pattern of hypospermatogenesis was characterized by triangular clusters "Sertoli cell only" tubules (no germ cells present) beneath the testicular capsule. Six- to eight-month-old dogs had a greater incidence of germ cell depletion and significantly lower testicular weights than older dogs. Retained spermatids, multinucleate giant cells, and swollen spermatocytes of minimal severity were common findings in all age groups. Germ cell vacuolation, apoptotic cells, and dilated tubules occurred in 3.7% to 16% of dogs.
P59 The Role of Cyclooxygenases in the EyePfizer Global Research & Development, Ann Arbor, MI, United States Cyclooxygenase isoenzymes (COX-1 and -2) catalyze the conversion of arachidonic acid to prostaglandins (PGs) and play a significant role in the health and disease of the eye. Experimental animal models of ocular diseases have been used to assess the effects of the selective COX inhibitors (COXIBs) and non-selective, non-steroidal anti-inflammatory (ns-NSAIDs) drugs, which are currently employed in the management of various ophthalmic conditions. This poster provides a review of the comparative expression of COX-1 and COX-2 in the eye under normal and pathological conditions, including the pathophysiologic role of PGs, and the effects of ns-NSAIDs and selective COX-2 inhibitors in the treatment of ophthalmic conditions.
P60 Thioacetamide-Induced Chronic Hepatotoxicity in BALB/c Mice: A Fibrosis ModelSchool of Veterinary Medicine, University of São Paulo, São Paulo, SP, Brazil Liver fibrosis is the common final pathway in most chronic liver diseases. Animal models such as knockout and transgenic mice provide means to study the fibrotic process. However, the experimental development of hepatic fibrosis in mice is more difficult than in rats. Therefore, we intended to improve liver fibrosis in mice using 4 different doses of thioacetamide (TAA). Forty BALB/c mice were injected i.p. with TAA (groups: 0.2, 0.1, 0.05, and 0.025 mg/g of body weight) twice weekly for 24 weeks. Liver was measured and metacarn-fixed liver samples were processed in order to quantify collagen volumetric proportion and to evaluate the cellular proliferation index. Data were analyzed by t-Student test (p < 0.05). TAA administration resulted in 20%, 50%, 30%, and 20% mortality after the first injection, respectively. Hepatosomatic index presented a statistical difference between groups 0.2 and 0.025 mg/g. Collagen volumetric proportion was, respectively, 20.5%, 13.7%, 14.5%, and 9.5%, in which only between groups 0.1 and 0.05 mg/g had no statistical difference. About 40% of animals that received 0.2 mg/g dose presented hepatic neoplasm. Hepatic proliferation index was, respectively, 0.73%, 0.14%, 0.18%, and 0.36%. These results demonstrate that the best model of hepatic fibrosis in mice was obtained with 0.1 or 0.05 mg/g doses of TAA, because the content of collagen that was reached was satisfactory and there was no tumoral development. The pathogenesis of liver fibrosis is only partly understood; therefore, the mice models are essential to further elucidate the mechanisms and for the development of new therapeutic strategies.
P61 A Systematic Assessment of Normal Tissue Expression of Mucosal Addressin Cell Adhesion Molecule-1 (MAdCAM-1) in Cynomolgus Macaque
1 Pfizer, Inc., Groton, CT, United States
Mucosal addressin cell adhesion molecule-1 (MAdCAM-1) is the predominant ligand for the integrin
P62 An In Vitro Study on Effects of Endosulfan Residues on Primary Cell Culture of Buffalo Lymphocytes
1 Preclinical Studies, School of Veterinary Medicine, St George’s University, St George’s, Grenada and In a study, residues of endosulfan varied from 0.018 to 1.550 µg/g in the meat of buffalo in India. Hence the present study was conducted to evaluate the in vitro effects of this pesticide on buffalo lymphocytes. For this purpose lymphocytes, collected from peripheral blood of buffalo, were divided into groups I, II, III, IV, and V and suspended in the 0, 0.5, 1.5, 2.5, and 3.5 ppm concentration of endosulfan, respectively, for one hour at 37°C. The cells after washing were incubated at 37°C with 5% CO2 for 68 hours. The cell culture fluid was collected for determination of NO concentration, IL-1, IL-2, and IgG levels and by MTT dye assay lymphocyte blastogenesis for B- and T-cells. Levels of the IL-1, IL-2, and IgG in the cell culture fluid were measured by ELISA by using the respective monoclonal antibodies. In vitro exposure of endosulfan to buffalo lymphocytes led to a decrease in B-cell blastogenesis of 76.17%, 49.32%, 34.22%, and 17.44% at concentrations of 3.5 ppm, 2.5 ppm, 1.5 ppm, and 0.5 ppm. The percent of decrease in T-cell blastogenesis was 71.30%, 49.32%, 34.22%, and 17.44% due to concentrations of 3.5 ppm, 2.5 ppm, 1.5 ppm, and 0.5 ppm of endosulfan exposure, respectively. IL-1 and IL-2 production in lymphocyte culture medium was significantly decreased with 1.5 ppm or higher concentrations of exposure to endosulfan. The mean ELISA value for IgG was 3.859, 1.632, 1.824, 2.249, 3.256, and 3.567 for groups I, II, III, IV, V, and VI, respectively, indicating that IgG production was significantly decreased by the 1.5 ppm or higher concentrations of exposure to endosulfan. These data suggest that very low concentrations of the pesticide as a residue in food items are potentially immunotoxic.
P63 Avian Primary Lymphocyte Culture as a Model for In Vitro Immunopathological Studies of Lindane
1 School of Veterinary Medicine, St. George’s University, St. George’s, Grenada and The search for valid alternatives to animal testing has been an area of research for many years. In vitro, cellular models can be useful for immunopathological research and may have predictive potential in the assessment of toxicity and, consequently, safety evaluation. In the present study, immunopathological effects of lindane were observed in the avian splenic lymphocyte cell culture. Lymphocytes were collected by mincing the spleen in RPMI – 1640 medium and subsequent washing. The cells were treated with lindane (suspended in RPMI – 1640) for 30-, 60-, and 90-minute using doses of Nx10–2, Nx10–3, Nx10–4, Nx10–5, Nx10–6, and Nx10–7 (where N = NOEL dose). After the exposure, observations were made to determine T-cell blastogenesis using MTT dye as indicator and Con A as mitogen, B-cell blastogenesis using MTT dye as indicator and LPS as mitogen, cytokine production (IL-1 and IL-2) using ELISA and monoclonal markers and apoptosis by immunoperoxidase and electron microscopy (only 60-min exposure). Following exposure, there was a decrease of 80.35%, 56.37%, and 39.79% in T-cell blastogenesis (MTT dye as indicator and Con A as mitogen), over the control, by 30-, 60-, and 90-minute exposure to Nx10–2 dilution of lindane and 76.58%, 74.10%, and 73.52% decrease in B-cell blastogenesis at the same concentration and time. Similarly a significant decrease in levels of IL-1 and IL-2 was also found. Smear of cells stained by immunoperoxidase technique for phosphatidylserine showed cells positive for apoptosis. Electron microscopic studies also confirmed the apoptosis in lindane-exposed lymphocytes. These results can be compared to the in vivo studies to formulate a viable cellular model for lindane immunopathological studies.
P64 Barbiturate Euthanasia Solution Induced Tissue Artifact in Nonhuman Primates
1 Southwest National Primate Research Center at the Southwest Foundation for Biomedical Research, San Antonio, TX, United States Barbiturates are known to induce necrosis, edema, congestion, hemorrhage, hemolysis, and emphysema via endothelial damage, smooth muscle relaxation, decreased calcium transport, and direct caustic effects in a variety of laboratory animals. There has been only one report of caustic effects in a nonhuman primate, Macaca speciosa. To further examine barbiturate euthanasia–induced tissue damage in nonhuman primates, 117 nonhuman primates representing seven species were evaluated. Animals were euthanized for humane and research reasons by various vascular routes with Euthasol®, Sodium Pentobarbital, Fatal Plus®, Beuthanasia D®, or Euthanasia 5®. The lungs and livers were examined histologically and blindly graded for hemolysis, vascular damage, edema, and necrosis. Four nonhuman primates that died of acute natural death served as controls. The degree of tissue damage was analyzed statistically for differences due to agent, age, sex, dose, and injection route. Artifacts observed in the tissues of the nonhuman primates in this study are the same as what has been reported in other laboratory animal species. Tissue damage severity was found to be directly related to dose and the intracardiac injection route. When the recommended dose of any of the agents was used, tissue damage was generally reduced, minimal, or undetectable. We recommend using the lowest dose of barbiturate euthanasia agent possible and that it be injected as far from the target study tissue as possible. These recommendations will help ensure that the quality of the tissue obtained from studies in nonhuman primates is compatible with project goals and that euthanasia procedures are properly performed.
P65 Comparison of Current Technologies for Capturing Photomicrographs for Journal SubmissionNational Institute of Environmental Health Sciences, Research Triangle Park, NC, United States The majority of journal submissions of photomicrographic images that are used to document scientific findings generally come from either 35mm film, digital image capturing, or digital slide scanning. Traditionally, 35mm cameras were attached to light microscopes to capture scientific images. These high-resolution images were developed and submitted to journals for scanning and placement in the accompanying manuscript. The scanning process tends to reduce image quality and resolution. With the advent of the digital microscope camera, the 35mm camera was replaced and investigators were able to produce digital images directly from their light microscopes. These images allowed for easier incorporation into the manuscript. When using digital cameras, care must be taken with slide preparation, Kohler illumination, white balancing, and focus. The digital slide scanner produces a high-resolution image directly from the slide with magnification up to 40x. Because of the amount of resolution involved, the file sizes are quite large and the scanners are more expensive than the previously discussed options. Examples of images captured via film, digital microscope camera, and digital slide scanning will be presented along with the pros and cons of these different technologies. We will examine these current technologies in the areas of ease of use, initial cost, time, resources, and output.
P66 Comparison of Three PPAR-alpha Selective Agonists in a Rat Toxicity Study Using Targeted Gene Induction as a Pharmacodynamic Surrogate to Estimate Therapeutic Safety Margins
1 Bristol-Myers Squibb Company, New Brunswick, NJ, United States
A 2-week rat toxicity study was expanded to compare the safety profiles of 3 peroxisome proliferator activated receptor alpha (PPAR
P67 Diethylnitrosamine (DEN)-Induced Initial DNA Damage Is Associated with Superoxide Formation, Up-Regulation of MDM2 and Down-Regulation of Constitutive Androstane ReceptorFaculty of Veterinary Medicine and Animal Sciences, University of São Paulo, São Paulo, SP, Brazil Activation and metabolism of DEN can produce reactive oxygen species (ROS) that cause DNA adduct formation and, consequently, mutations in cancer-related genes. In this study we have characterized the acute response in the liver to an alkylating carcinogen and showed alterations related to severe DNA damage. We used 25 two-month-old BALB/c female mice injected with DEN (160 µg/kg, i.p., saline) or saline (i.p.). Four hours post-dosing, the mice were euthanatized. The livers were collected, weighed, and snap frozen for DNA ladder assay, Catalase (CAT), Gluthatione reductase (GRED), and Superoxide Dismutase (SOD) activity, and real-time PCR analysis of these genes plus MDM2, CAR, and Gluthatione peroxidase (GPX). Serum samples were collected for ALT and GGT analysis. After 4 hours, no change in body and liver weight was noted but increases in GGT (p < 0.0001) and DNA damage were evident. On the other hand, total protein (p = 0.0007), cholesterol (p = 0.0211), triglycerides (p < 0.0001), and urea (p = 0.0206) were decreased in serum after DEN administration. SOD activity was increased (p = 0.0032), but its mRNA was 0.62-fold down-regulated. CAT, GRED, and GPX activity were not changed; however, expressions were down-regulated (respectively, 0.21-, 0.19-, and 0.30-fold). Interestingly, we detected a 21-fold MDM2 up-regulation and a 0.24-fold CAR down-regulation. We conclude that metabolism of DEN specifically increases the superoxide formation consequently leading to DNA damage, which is associated with decreased CAR expression resulting in diminished drug-metabolizing gene expression. It is known that ATM kinase activation can occur after DNA damage and stabilize p53 in the nucleus, leading to a transcriptional up-regulation of MDM2.
P68 Evaluation of Organ Weights for Rodent and Non-Rodent Toxicity Studies: A Review of Regulatory Guidelines and a Survey of Current Practices
1 Merck Research Laboratories, West Point, PA, United States Regulatory guidance documents describing organ weighing practices are generally available; however, they differ somewhat dependent on industry type and regulatory agency. The Society of Toxicologic Pathology convened a working group to evaluate current practices regarding organ weights in toxicology studies. A survey was distributed to pharmaceutical, veterinary, chemical, food/nutritional, and consumer product companies in Europe, North America, and Japan. While questionnaire respondents unanimously stated that organ weights were a good screening tool to identify treatment-related effects, opinions varied as to which organ weights are most valuable. The liver, kidneys, and testes were commonly weighed and most often considered useful by most respondents. Other organs that were commonly weighed included brain, adrenal glands, ovaries, thyroid glands, uterus, heart, and spleen. Organs such as lungs, lymph nodes, and other sex organs were weighed infrequently in routine studies, but were often weighed in specialized studies such as inhalation, immunotoxicity, and reproduction studies. Organ to body weight ratios were commonly calculated and were considered more useful when body weights varied between groups. Organ to brain weight ratios were calculated by most North American companies, but rarely according to respondents representing veterinary product or European companies. Statistical analyses were generally performed by most respondents. Pathologists performed interpretation of organ weight data for the majority of the industries.
P69 In Vitro Evaluation of Diuron as a Possible DNA Crosslink Inducer by the Comet AssayTOXICAM, UNESP Medical School, Botucatu, São Paulo, SP, Brazil Diuron, an urea-derived herbicide, is carcinogenic to the male Wistar rat’s urinary bladder but has questionable genotoxic potential. The comet assay (Single Cell Gel Eletrophoresis Assay) is a well-established genotoxicity assay for in vitro and in vivo testing of chemicals. The technique allows rapid, simple, and sensitive detection of DNA damage at a single cell level. In a previous in vivo study using the standard comet assay protocol, Diuron did not induce DNA damage in rat urothelial and peripheral blood cells. However, the molecular structure of Diuron has two potential reactive radicals, indicating the possibility of crosslink induction, which is not readily detected by the standard comet assay. In contrast to other different DNA lesions, crosslinks are the only known DNA modification that cause reduction of eletrophoretic migration of DNA fragments. Thus, the possibility of Diuron to induce crosslinks was evaluated by an in vitro modified comet assay protocol that used a combined treatment with Diuron and methyl-methanesulphonate (MMS), a recognized chemical agent that allows DNA migration in the comet assay. CHO cells were treated with Diuron for 3 hours and subsequently exposed to MMS for 1 hour to determine a possible reduction in MMS-induced DNA migration. Based on its water solubility (40 µg/mL), three concentrations of Diuron were studied: 50, 25, and 12.5 µg/mL. Tripan blue exclusion assay conducted immediately after Diuron treatment indicated cell viability higher than 94% for all treatments. Our results indicate that Diuron concentrations tested did not reduce DNA migration induced by MMS. Supported by TOXI-CAM and CNPq.
P70 Incidence of Spontaneous Background Microscopic Observations in 34 Beagle Dogs: A Cross-Sectional StudyWyeth Research, Chazy, NY, United States Spontaneous microscopic observations were assessed in 1.8 to 7 years old males (n = 11) and females (n = 23) purpose-bred adult beagle dogs in an effort to better distinguish spontaneous from toxic changes. All animals were provided by a commercial breeder (Marshall BioResources) between the age of 5 and 10 months and were kept for long-term maintenance and/or routine staff training with canines. Mononuclear cell inflammation, mineralization, and cysts in various tissues were the most common findings and usually had comparable incidence in males and females. Mononuclear cell inflammation occurred more frequently in the stomach (97%), liver (88%), gallbladder (62%), and prostate (55%). Mineralization occurred frequently in the aorta and kidneys. In the aorta, mineralization was evident in the tunica media of the proximal aorta (65%). Renal mineralization only occurred within the papillary tubules (97%). Cysts were present in the thymus, pituitary gland, and ovaries. Within the thymus, cysts (91%) were associated with physiological age-related involution (100%). Pituitary gland cysts had a higher incidence in the pars intermedia (74%) than in the pars distalis (47%). Ovarian cysts were more frequently of follicular (26%) origin. Some lesions were less common but of higher interest in terms of historical data. These included hepatic nodular hyperplasia (5.3-year-old female) and hepatocellular adenoma (5.4-year-old female), thyroid follicular epithelial hyperplasia (7-year-old male), and renal tubular epithelial hyperplasia (3.2-year-old male). These microscopic observations emphasize the utility of consistently recorded and readily accessible archived control data when attempting to differentiate background spontaneous changes from compound effect.
P71 Incidences and Range of Spontaneous Lesions in Control Laboratory Beagle Dogs Used in Toxicity StudiesCharles River Laboratories, Edinburgh, United Kingdom A retrospective study was performed to determine the incidences and range of spontaneously arising pathological findings in control Beagle dogs. A total of 402 dogs (7 to 20 months) from 56 toxicity studies evaluated between 2002 and 2007, with equal numbers of male and female animals, and full tissue lists were included in the study. Inflammatory cell infiltration was the most commonly recorded finding, with the liver (38%), lung (22%), salivary glands (16%), and kidneys (7%) the most affected organs. The second most commonly encountered finding was erythrophagocytosis of the mesenteric lymph nodes (30%). Inflammatory lesions were mostly encountered in the lung, with chronic interstitial inflammation (alveolar septal fibrosis) (17.7%), bronchopneumonia (15.4%), and pleural fibrosis (9%) being the most common. The most common degenerative change was mineralization, with renal tubules (22%), gastric mucosa (10%), and aortic wall (8%) the most affected, whereas adrenal cortical vacuolation (13%) in females was second. Pituitary cysts (17%) and valvular hematocysts of the heart (9%) were the most common congenital lesions, whereas viral papillomas (2%) and histiocytoma (0.5%) of the skin were the only neoplastic findings recorded. There were sex differences in the incidences of adrenal cortical vacuolation, tubular epithelial vacuolation of kidneys (lipidosis), mineralization of gastric mucosa, and valvular hematocyst. Factors such as the source and genetic background of animals, study types, management and housing of animals, and variations in recording levels among study pathologists have to be considered in the interpretation of the results.
P72 Metabolomic Response Profile for Sham Surgery and Radiation in Mice
1 Metabolon, Inc., Durham, NC, United States and We present a metabolomic analysis of the biochemical responses of an animal to a toxin in which various aspects of the mechanisms of toxicity may be distinguished from one another. The long-term objective of this study is to identify biomarkers for the induction of potentially lethal hepatic radiation injury, which is induced after whole liver irradiation in cancer patients. We determined the patterns of small molecules and biochemical markers after whole liver irradiation in mice. There were four treatment groups: untreated mice; sham surgery-treated mice; sham surgery-treated mice with livers exposed to either low (10 gray [gy]) or high radiation (50 gy). Blood samples were taken at 24h and analyzed using GC-MS and LC-MS. Relative concentrations of over 150 small molecules were determined by matching spectral data from known compounds. Statistical analysis allowed the classification of mice between the four treatment groups through the identification of two groups of ten biochemicals. The most significant of these included citric acid for one group and 1,5-diaminopentane for another, which distinguished mice for radiation effects and surgery, respectively. Metabolomics offers a unique understanding of biochemical changes induced by the introduction of toxic agents, while differentiating the effects of surgery.
P73 Metabolomics Analysis of Plasma and Urine in Rats Treated with Fenofibrate
1 Metabolon, Inc., Durham, NC, United States and
Fenofibrate, a peroxisome proliferator-activated receptor-
P74 Publication Images: To Adjust or Not to Adjust
1 National Institute of Environmental Health Sciences, Research Triangle Park, NC, United States and Photomicrographic images that document scientific findings submitted to Toxicologic Pathology range in quality from the good and the bad, to the impossible. Some of the most common problems with manuscripts submitted to Toxicologic Pathology are uneven illumination, out-of-focus photomicrographs, overexposed immunohistochemistry, and inappropriate labeling. With the widespread access and availability to digital imaging equipment and image manipulation software, it is relatively easy to alter images that document scientific findings. With a focus on photomicrographs, we will examine the ethics of digital image manipulations. Some changes, including cropping or intensity adjustments to the entire image, are usually acceptable. However, use of software "filters," color adjustments, interpolation, and cloning are generally inappropriate and/or unethical. Generic guidelines for acceptable image adjustments and suggested documentation for these photomicrographs will be presented.
P75 Society of Toxicologic Pathology Position on Organ Weight Recommendations for Toxicology Studies
1 Albert Einstein College of Medicine, Bronx, NY, United States The evaluation of organ weights in toxicology studies is an integral component in the assessment of pharmaceuticals, chemicals, and medical devices. The Society of Toxicologic Pathology (STP) has generated recommendations for weighing organs in GLP general toxicology studies (lasting from 7 days to 1 year) based both on scientific merit and practicality. The STP advises that liver, heart, kidneys, brain, testes, and adrenal glands be weighed in all multi-dose general toxicology studies for all species; thyroid gland and pituitary gland weights are also advocated for all species except mice. Spleen and thymus should be weighed in all rodent studies and should be considered in non-rodent studies. Reproductive organs are best evaluated in sexually mature animals. Epididymal and prostate glands should be weighed routinely in rodent general toxicology studies, but should be optional in non-rodent studies because high animal-to-animal variability, inclusion of immature animals, and small numbers of animals complicate interpretations. Weighing of other organs including female reproductive organs should be considered on a case-by-case basis. Organ weights are not recommended for any carcinogenicity studies including the alternative 6-month mouse bioassays. Regardless of the study type or organs evaluated, organ weight changes must be evaluated within the context of the compound class, mechanism of action, and the entire data set for that study.
P76 A Stable Transgenic Zebrafish for Developmental Toxicology by Aryl Hydrocarbon Receptor Binding ToxicantsSeoul National University, Seoul, South Korea We first established a transgenic zebrafish line carrying a plasmid (phAhRE-EGFP) for detecting developmental toxicities caused by aryl hydrocarbon receptor (AhR) binding toxicants. The plasmid contains the 5’ human cytochrome P4501A1 (CYP1A1) upstream sequence, which AhR ligands target, and enhanced green fluorescent protein (EGFP) gene, an accurate and quick, inducible reporter. To evaluate this system, we treated embryos of wild-type and the transgenic fish with four well-known AhR binding toxicants: 3,3’,4,4’,5-pentachlorobiphenyl (PCB126); 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD); 3-methylcholanthrene (3-MC); and β-naphthoflavone (β-NF). These toxicants induced a dose-dependent increase in morphologic disruption, indicating toxicity at early life-stages. The transgenic zebrafish were sensitive enough to these toxicants to express the CYP1A1 regulated EGFP. Moreover, fluorescence-based quantification of the reporter gene was consistent with dose-dependent toxicity of the four model toxicants. In conclusion, the stable transgenic line will allow for extremely rapid and reproducible toxicological profiling of early life-stage embryo development in both aquatic organisms and humans.
P77 The Effect of the VivoMetrics® LifeShirtTM System on Mean Arterial Blood Pressure, Heart Rate, and Body Temperature in Unrestrained Cynomolgus MonkeysCharles River, Sparks, NV, United States The VivoMetrics® LifeShirtTM System (Ventura, CA) permits the potential to measure functional parameters of the respiratory system concomitant with mean arterial blood pressure (MAP), heart rate (HR), and body temperature (BT) in telemetry instrumented cynomolgus monkeys. We measured the MAP, HR, and BT under the following conditions: 1) LifeShirtTM + protective jacket, 2) LifeShirtTM + protective jacket + recorder, 3) protective jacket + simulated recorder weight (500 mL saline bag), 4) protective jacket, and 5) no apparatus (control). Compared to control, treatments 1 & 2 had similar effects of higher MAP (40 mm Hg [lights-on] to 55 mm Hg [lights-off]) and HR (25 beats per minute [bpm] [lights on] to 60 bpm [lights-off] plus loss of their expected reduction during lights-off). Compared to control, treatments 3 and 4 had similar effects of higher MAP (20 mm Hg [lights-on] to 30 mm Hg [lights-off]) and loss of MAP reduction during lights-off. Similarly, treatments 3 and 4 resulted in higher HR (0 to 10 bpm [lights-on]) plus attenuated HR reduction (~30 bpm) during lights-off. Compared to control, treatments 1 through 4 resulted in lower BT (0.5°C to 1.0°C) during lights-on and treatments 1 and 2 attenuated the BT reduction during lights-off. Results indicate 1) MAP, HR, and BT are affected by concomitant use of the LifeShirtTM with protective jacket, and 2) LifeShirtTM + protective jacket had the greatest effect irrespective of recorder weight.
P78 The Value of Automated Cellular Imaging on Tissue Microarrays in the Drug Discovery ProcessAstraZeneca, Alderley Park, United Kingdom Tissue Microarray (TMA) technology is currently being utilized as a complimentary histological tool for both normal target distribution and evaluation of pharmacodynamic biomarkers in support of the drug discovery process. The Automated Cellular Imaging System (ACIS - Clarient Chromavision) provides a rapid, precise approach to the quantitative analysis of immunohistochemically localized protein within TMAs. This system utilizes color space analysis to determine the percentage of protein localization, in relation to a selected counterstained tissue component area, and relative optical intensity as an indication of protein density in TMAs. We describe the use of the ACIS under two situations (1) identification of novel target molecule distribution in normal animal TMAs and (2) analysis of a panel of immunohistochemical biomarkers to determine mode of action of novel anti-cancer drugs in xenograft TMAs. In the first case, the approach taken was to utilize a two-tiered analysis, identifying that protein expression was present in any particular tissue core and then reviewing those cores for the specific cellular distribution of protein. In the second example, immunohistochemistry on TMA samples was used to determine the pharmacological effects of small molecule inhibitors on molecular targets and linked pathways, including apoptosis and endoreduplication. TMAs were also stained by the histochemical Blue Feulgen method for ploidy evaluation on the ACIS. These analyses have indicated the value of using a combination of Chromavision ACIS and histological analysis of TMAs for both novel target distribution in drug discovery and in predicting the efficacy of cyclin and mitosis inhibitors in preclinical models.
P79 Uncommon Spontaneous Findings in Laboratory BeaglesHuntingdon Life Sciences, Huntingdon, Cambridgeshire, United Kingdom Knowledge of background findings is indispensable in assessing the significance of unusual histopathological changes, notably in dog and primate studies where the numbers of animals per group are small. Some unusual incidental findings that were recently noted in beagles at our laboratory are described here. These include a coloboma of the optic disc, Renaut bodies in the sciatic nerve, a cystic developmental anomaly in the testis, endometrial hyperplasia in the uterus, and spontaneous extramural lymphocytic arteritis in the coronary vessels of the heart. Reports of these lesions in clinical cases and laboratory beagles are rare, with limited descriptions of their histopathological features in the literature. This poster illustrates and describes the histopathological features of these spontaneous findings, briefly discussing their significance and pathogenesis.
P80 Use of Human Liver Slices to Evaluate and Compare In Vitro Toxicity of Biologic Compounds
1 Biogen Idec, Cambridge, MA, United States and In vitro cultured, precision cut human liver slices have been used for many years to assess hepatotoxicity with a variety of drugs and chemicals. The purpose of this study was to investigate the potential hepatotoxicity of a biologic drug candidate with reactivity limited to human and chimpanzees, and compare it to a potential surrogate homologue molecule with similar activity and broader species cross-reactivity.
Liver slices were treated with multiple dilutions of the test compounds, TNF-
Allyl alcohol–treated positive control showed significant changes in K+, ATP, LDH, ALT, and AST, as expected. Negative control samples showed good viability and no indication of hepatocellular damage through the 24-hour time point. In the present case, there were no observable dose- or time-dependent effects on ATP content, K+ gradient, or liver enzymes that were related to treatment with TNF-
P81 Use of Image Analysis Techniques to Evaluate Neuronal Necrosis in a Kainic Acid Model of Mesial Temporal Lobe Epilepsy
1 Lilly Research Laboratories, Greenfield, IN, United States This study evaluated different methods of quantification of neuronal necrosis in a kainic acid rodent model of mesial temporal lobe epilepsy, using digital whole slide images. After evaluation of various software packages for ease of application and available algorithms for image analysis, two main procedures for the identification and quantification of neuronal necrosis were compared. Evaluations were performed on digitized images (Aperio® T2) of rostral hippocampal sections in rats. The first procedure involved a manual count of necrotic and total neurons in the Cornu Ammonis (CA) 1 region of a section of hippocampus delineated using tools available with the scanning software. This technique was relatively simple, correlated well to semi-quantitative assessments provided by independent review from a pathologist, but required review of each individual slide. The second procedure focused on custom-created algorithmic methods for automated necrotic neuron detection using Matlab® and its associated image processing toolbox. Automated identification of the CA1-4 and dentate gyrus portions of the hippocampus was achieved using segmentation techniques. However, identification of the necrotic regions proved to be difficult due to the overlap of color spectrum between normal and abnormal neurons, and lack of visible cellular definition. Through further refinements of the custom algorithm, for example, use of blurring and filtering techniques, we hope to isolate the necrotic cells for positive identification. Development of an automated tool is still under way. In conclusion, this study identified critical features unique to whole slide digital image capture and will provide the framework for further development and refinement of an automated tool.
Toxicologic Pathology, Vol. 36, No. 1,
149-176 (2008)
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-H2AX foci, total 
and –β, progesterone (PR) receptor, androgen receptor (AR), and aromatase cytochrome P450 in the skin of Göttingen minipigs. Eighteen Göttingen minipigs (3 animals/sex/age group: 3–4, 6–8, and 12–15 months) were examined. Skin samples were obtained from designated cranial and caudal regions of the dorsum of each pig. These were prepared for microscopic examination using routine H&E, trichrome, or immunohistochemical stain. The thickness of the stratum corneum, epidermis, and dermis were measured microscopically. Older pigs had a thicker dermis than younger pigs. Older females had a thinner epidermis. In older pigs, the cranial dermis was thicker than the caudal dermis. Hair follicles, sebaceous glands, keratinocytes, and dermal fibroblasts were variably positive for ER