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AbstractsP1 "Instantaneous" Heart Rate Variability (HRV) in Awake Dogs: A Sensitive and Specific Assay for Safety PharmacologyBioTechPlex Corporation, Elk Grove Village, United States Due to the ever changing nature of biological signals, we hypothesized that extraction of autonomic indexes from the modulation of the ECG using time-frequency analysis rather than using Fourier analysis would enable more rapid assessment of autonomic function with greater specificity and higher sensitivity. We have developed a system, CardioDataPadTM, to extract the powers of the modulations of the R-R intervals within the high frequency (HF) (2.5-6s) and low frequency (LF) (6-25s) bands using non-stationary time-frequency analysis at 30 second intervals as well as using Fourier analysis at 5 minute intervals. The ECG, sampled at 500 Hz, was measured in a cohort of 8 beagle dogs (10–12 kg) which previously had their vagosympathetic trunks elevated and placed in skin tubes. In 6 unanesthetized dogs, 1–4 ml of lidocaine administered to the skin tubes decreased the power of the HF component 86 times. Administration of the central nicotinic receptors antagonist mecamylamine (1 to 10 mg/kg i.v.), caused a 10 fold inhibition of the LF and HF (n = 7). The short-term anesthetic, propofol, administered i.v. to 6 dogs caused a suppression HF to 1/200 of the baseline values. At this markedly reduced level of autonomic tone, the LF was nearly 20 times that of the HF. These data indicate that evaluation of autonomic tone for safety pharmacology should not be assessed in anesthetized animals and that the time-frequency methods, inherent in CardioDataPad TM, have a minimum of 10 times the sensitivity and 4 times the specificity compared to conventional HRV analysis.
P2 2-Butoxyethanol-Female Rat Model of Hemolysis and Disseminated Thrombosis: X-Ray Characterization of Osteonecrosis and Growth Plate SuppressionNational Institute of Environmental Health Sciences, Research Triangle Park, NC, United States We recently proposed a chemically-induced rat model for human hemolytic disorders associated with thrombosis. The objective of the present investigation was to apply a non-invasive, high-resolution x-ray analysis to characterize the protracted bone damage associated with this model, and to validate it by histopathology. Groups of female Fischer 344 rats were given 0 mg, 250 mg, or 300 mg of 2-butoxyethanol/kg body weight daily for four consecutive days. Groups were then sacrificed two hours or 26 days after the final treatment. The treated animals had a red discoloration on the distal tail. Animals sacrificed two hours after the final treatment were examined histologically and disseminated thrombosis and infarction was seen in multiple organs, including bones. Faxitron radiography was used to take images of selected bones of rats sacrificed in 30 days. Premature thinning of the growth plate was seen in the calcaneus, lumbar and coccygeal vertebrae, femur, and ilium from the treated animals. An increased radiolucency in the diaphysis of the femur was also seen in all treated animals. The bones were then examined histologically, showing a range of changes including loss or damage to growth plates and necrosis of cortical bone. No thrombi were seen in the animals examined in 30 days, but there were bone and growth plate changes consistent with prior ischemia. The Faxitron proved to be an excellent non-invasive tool that can be used in future studies with this animal model to examine treatment modalities for the chronic effects of human thrombotic disorders.
P3 An Adaptable Grading System and Criteria for Cardiovascular Device EvaluationEPL, Inc., Sterling, VA, United States A grading system and examples of some of the severity criteria used for evaluating pathologic changes associated with cardiovascular devices are presented. The system can be tailored to the experimental design and the tissue of interest including non cardiovascular tissues. The grading system is based on a 1 through 4 scoring scheme but can be changed to other numerical score systems. The presented system also covers the commonly expected changes that may occur after device implantation and/or manipulation of the host tissues and includes types of inflammatory cell infiltrates, fibroblast infiltration, fibrosis, subintimal or pseudointimal proliferation, neovascularization, congestion, elastin degeneration, smooth muscle myofiber proliferation, calcification/mineralization, acelluarity/necrosis, endothelial cell proliferation, hemorrhage and fibrin accumulation. Other changes of specific interest can be incorporated into the system such as the results of immunohistochemical staining. Each of these changes can also be localized to different aspects of the vessel or heart (aorta, vena cava, valve leaflet, endocardium, et cetera). The grading system can also be modified to address changes to the device such as ingrowth of host tissue and/or degradation of the device depending on what is appropriate to the experimental design. Examples of many of these changes are presented.
P4 Cardiac Injury from Long-Term Episodic Exposure to Particulate Matter (PM): Soluble Components or Solid Particles?
1 U.S. Environmental Protection Agency, NHEERL, Research Triangle Park, NC, United States Long-term exposure to PM has been associated with cardiac injury in rats. The purpose of this study was to investigate if cardiac injury was due to soluble metals (i.e., zinc), insoluble PM, or pulmonary injury/inflammation. Male Wistar Kyoto rats (n = 8) were exposed intratracheally (IT), once/wk x 8 wks to either saline, Mount St. Helen’s ash (MSH, no soluble metals)—2.3 mg/kg/wk, environmental combustion particles (PM, zinc as soluble metal)—1.15 or 2.3 mg/kg/wk, the saline-leachable fraction of PM (PM/L)—2.3 mg/kg/wk or ZnSO4 (Zn)—33.4 µg/kg/wk (equivalent soluble zinc in PM). Another group of rats was exposed to half the concentration of all components above but for 16 wks. All IT exposures, except saline, caused marked lung inflammation including MSH. Lavage fluid protein, albumin, and LDH activity also increased in all groups relative to saline. PM high dose demonstrated the greatest pulmonary effects. No changes were apparent among the hematological parameters. Histologically, minimal to no lesions were noted in controls. Mild to moderate myocardial lesions were noted in all other groups including MSH; however, the mean severity was highest in PM high dose group. Subepicardial and random myocardial lesions were characterized by myocardial degeneration, inflammation, and fibrosis. In summary, long-term episodic IT exposure to insoluble PM or soluble zinc causes persistent pulmonary inflammation/injury as well as varying degrees of cardiac lesions. These findings support the hypothesis that both soluble and insoluble components may cause cardiac injury. (Does not reflect U.S. EPA policy).
P5 Cerebrovascular Localization of COX-1, COX-2, and iNOS in Humans with Stroke LesionsPfizer Global Research and Development, Ann Arbor, MI, United States Focal cerebral ischemia is associated with a marked inflammatory reaction that contributes to the evolution and progression of brain tissue injury. Studies employing anti-inflammatory compounds and transgenic mouse models have suggested that both cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) mediate the deleterious effects of ischemic brain injury. In order to evaluate the expression profiles of these enzymes in the normal and post-ischemic human brain and associated vasculature we evaluated the tissues from 11 patients with a morphologic diagnosis of cerebrovascular disease or cerebrovascular accident (CVA) for the expression of COX-1, COX-2, and iNOS by both immunohistochemistry and in situ hybridization. Brain sections from corresponding regions of six normal patients served as controls. COX-1, COX-2 and iNOS were all present in the normal brain parenchyma. However, in the infarcted brains, an increase in iNOS and COX-2 but not COX-1 expression was observed. Increased COX-2 and iNOS expression was primarily seen in the parenchyma undergoing ischemic lesions. Vascular expression of COX-2 was seen in only one case and of iNOS in 3 cases. Our data demonstrate the up-regulation of both iNOS and COX-2 in ischemic parenchyma but not vasculature and provide support for the hypothesis that iNOS and COX-2 may contribute to the progression of post-ischemic cerebral injury via local cytotoxic rather than cerebrovascular mechanisms.
P6 Chemical-Induced Atrial Thrombosis in Rodent NTP Studies: Morphological Aspects and Potential Mechanisms
1 National Institute of Environmental Health Sciences, Research Triangle Park, NC, United States and Cardiac thrombosis, especially atrial thrombosis, accounts for more deaths in the world than any other disease, and major conditions in which thrombosis plays a principal role include several cardiovascular diseases, such as atrial fibrillation, myocardial infarction and stroke. Results from the induction of chemical thrombosis in rodents are useful to identify substances in our environment that may contribute to cardiac thrombosis. We evaluated atrial thrombosis induced by chemical exposures in rodents, compared it to similarly induced lesions reported in the literature, from the results of more than 500 chemicals in National Toxicology Program (NTP) database, and summarized a putative pathogenesis. The incidences of atrial thrombosis were increased in high-dosed groups in the NTP rodent studies of 13 compounds: 2-butoxyethanol, C.I. Direct Blue 15, bis(2-chloroethoxy)methane, diazoaminobenzene, diethanolamine, 3,3'-dimethyl-benzidine dihydrochloride, hexachloroethane, isobutene, methyleugenol, oxazepam, C.I. Pigment Red 23, C.I. Acid Red 114, and 4,4'-thiobis(6-t-butyl-m-cresol). The left atrium was the main localization in spontaneous and chemically-induced thrombosis. The literature survey suggested that chemical-induced atrial thrombosis might be closely related to myocardial injury, endothelial injury, circulatory stasis and/or hypercoagulability. Impaired atrial mechanical activity, such as atrial fibrillation, could cause stasis of blood within the left atrial appendage, contributing to left atrial thrombosis.
P7 Chemical-Induced Protection in Red Blood CellsUniversity of Louisiana at Monroe, Monroe, LA, United States A priming dose of either butoxyethanol (BE, 500-mg/kg, po) or phenyl hydrazine (PH, 125 mg/kg, po) leads to survivable hemolysis in female SD rats and protects against LD90 dose of BE (1500 g/kg, po) administered 7 or 14 days later, respectively. Half-life of butoxyacetic acid (BAA, hematotoxic metabolite of BE) is 3 h. Why high doses of BE lead to hemolysis beyond 4-half-lives of BAA, is unknown. We hypothesized that calpain (CAP), a death-protein released from lysed-red blood cells (RBCs) mediates the progression of BAA-initiated-hemolysis in rats treated with 500-mg BE/kg. Hemolysis was evident from 3 to 24 h. Higher CAP levels were found from 3 to 9 h, suggesting its role in the progression of injury. Incubation of RBCs with CAP (5.6 U) led to significant hemolysis by 1 h. After BE administration, rats recover due to production of new RBCs by 168 h. The new RBCs are resilient to hemolysis, leading to lower hemolysis on subsequent administration of LD90 dose of BE. We hypothesized that overexpression of calpastatin (CAST, an endogenous inhibitor of CAP), in the new RBCs will impart resistance to CAP-mediated degradation of RBCs. Higher CAST levels were found from 3 to 24 h. Contrary to our expectation, CAST overexpression was not seen in rats with induced reticulocytosis (1.4 mg PH/kg/d, 8 d, sc). Collectively, these results suggest that other death-proteins and their inhibitors may play a role in mediation of progression/regression of hemolytic injury.
P8 Effects of a c-Jun N-terminal kinase JNK inhibitor, SP600125, on Fumonisin B1 Induced Sphingolipid Alterations in Hepatic, Cardiac and Aortic Cell LinesUniversity of Illinois at Urbana-Champaign, Urbana, IL, United States
Fumonisin B1 (FB1) increases tissue sphinganine (Sa) and, to a lesser extent, sphingosine (So) concentrations and causes cardiovascular dysfunction and hepatocellular injury in vivo. We have previously shown that SP600125, a c-Jun N-terminal kinase (JNK) inhibitor, attenuates the TNF Supported, in part, by the Society of Toxicologic Pathology/Abbott Laboratories Clinical Research Award and International Life Sciences Institute.
P9 Endpoints in Transgenic Ras-Induced Cardiomyopathy Predicted by Means of Ultrasound Biomicroscopy in Mice Created Using a Conditional Reverse Tetracycline Transactivating Expression ConstructNational Cancer Institute, Bethesda, MD, United States Altered regulation of Ras, MAPK family members, and downstream transcription factors such as fos and myc have been associated with human cardiomyopathies. Transgenic mice engineered to over-express various mediators in the Ras-MAPK pathway from embryonic development, get lesions similar to human cardiomyopathy. A reverse tetracycline transactivating construct was used to express activated Ras in the hearts of 3 mice (MR) for 141 to 243 days after they were weaned. MR mice were compared to 6 wild-type littermates housed under identical conditions. All mice were examined periodically by ultrasound and congestive heart failure (CHF), evidenced by abdominal and pleural effusion, was documented in two of three MR mice. At endpoint, CHF mice had increased short axis biventricular diameter (cardiomegaly) and decreased left ventricular fractional shortening (myocontractility), compared to echocardiographic findings in wild-type mice. Necropsy corroborated clinical findings and heart weights were increased in the 2 MR mice with CHF. Histopathologically, there was variation in cardiac myofiber size, cardiocyte hypertrophy, loss of cross-striations, myofiber disarray, and interstitial fibrosis in all 3 MR mice. These findings confirm that conditionally targeted Ras expression results in lesions phenotypically similar to cardiomyopathies in human beings and in some other transgenic mouse models. Echocardiography serves as a suitable pathology adjunct for predicting study endpoints and may be useful for monitoring response to candidate new drug protocols in such systems. Future studies will include attempts to examine myocardial lesion pathogenesis in response to alternative Ras expression protocols.
P10 Ephendrine and Caffeine Cause Significant Mortality in 14-Week, but Not in 7-Week Old Fischer RatsNational Institute of Environmental Health Sciences, Research Triangle Park, NC, United States Human consumption of ephedrine and caffeine in dietary supplements has been associated with a number of adverse effects including changes in the electrocardiogram (ECG), myocardial infarction, hyperthermia, and in rare instances, death. The purpose of this study was to investigate the potential mechanisms associated with the cardiotoxicity of combined ephedrine and caffeine ingestion. Seven- and 14-week old Fischer 344 rats treated with ephedrine in combination with caffeine exhibited increases in heart rate (HR), temperature and QTc interval. Of the 14-week old rats treated with 25 mg/kg ephedrine plus 30 mg/kg caffeine, 57% died within 3–5 hours of treatment, while none of the similarly treated 7-week old rats nor any of the rats treated with vehicle died. One hour after treatment with this dose of ephedrine plus caffeine, 14-week old rats treated with this dose exhibited a larger increase in HR (as % increase over baseline) than 7-week old rats. Furthermore, the 14-week old rats that died had a higher HR and temperature than the 14 week old rats that lived. Histopathological studies suggested interstitial hemorrhage and myofiber necrosis in the 14-week old rats treated with the highest concentration of ephedrine and caffeine. This study showed enhanced susceptibility to ephedrine plus caffeine in 14-week old rats compared to 7-week old rats. The greater mortality in the 14-week old rats was associated with increases in body temperature, heart rate and myocardial necrosis.
P11 Evaluation of Glycosaminoglycans Content and 5-Hydroxytryptamine 2B Receptor in Rat Heart-Valves with Spontaneous Mitral Valvulopathy
1 GlaxoSmithKline, Research Triangle Park, NC, United States and 5-Hydroxytryptamine 2B receptor (5HT2BR) stimulation is known to cause fibroblast mitogenesis, and is likely to be associated with the development of valvulopathy in humans with carcinoid tumors and use of 5HT2BR agonists/anorxigens, such as fenfluramine, dexfenfluramine, ergot alkaloids (ergotamine and methysergide) and pergolide. Anorexigen-associated valvulopathy has been described as superficial "plaque-like" thickenings composed of distinctive glycosaminoglycans (GAGs)-rich fibromyxoid tissue on the surface of valvular leaflets in humans. Similar microscopic features have been observed in heart-valves with spontaneous mitral valvulopathy (SMV), also referred to as mitral endocardial myxomatous change in rats. Because of SMV’s microscopic resemblance to anorexigen-induced valvulopathy in humans, we investigate the presence and distribution of 5HT2BR and GAGs, and relate these to the presence of SMV in Sprague-Dawley (S-D) rats. Four to five-micron thick heart sections were cut from the formalin-fixed, paraffin-embedded tissue blocks and stained with Movat’s pentachrome for GAGs, and immunohistochemistry to detect tissue localization of 5HT2BR. Similar to anorexigen-induced valvulopathy in humans, valve-leaflets with SMV contained a greater amount of GAGs as compared to normal valves in S-D rats. Additionally, 5HT2BR-positive cells were substantially increased in valve-leaflets with SMV. Our S-D rat data showed increased GAGs content and number of 5HT2BR-positive cells in valve-leaflets with SMV, suggesting that 5HT2BR may play a pathogenetic role. However, additional research is needed to confirm the role of 5HT2BR in the pathogenesis of SMV in S-D rats.
P12 Heart and Thigh Muscle Effect in Mice Following Electroporation
1 Green Seasons Biotech Company, Tamshui, Taiwan and Electroporation, an alternative electric current device applied to inoculate in the injected site resulting in an explosion of inoculate to the peripheral tissue, was performed in thigh muscle of 42 young-adult BALB/c mice. Mice were divided into groups of 3 male and 3 female, each received a 0.2 ml inoculate of encoding protein prepared on viral plasmid-DNA at conc. of 0, 50, 150, or 400 ug/ml for intramuscular injection. Mice were sick in the next days and some died beginning at 2 day-post-exposure (dpe.). At 5 dpe., the study was terminated and a total of 26 survivals were submitted for pathological diagnosis. Significant histological observations were noted in the heart as necrosis with mineralization/cardiomyopathy (2 of 3 controls and 22/23 protein treatment mice), and skeletal muscle similar necrosis with mineralization/myopathy (1/3 control and 7/23 treatment mice). The incidences for the cardiomyopathy and myopathy in the control and treatment groups combined were 92%(24/26) and 35%(9/26), respectively. The cardiomyopathy was characterized by focal degeneration of myocardium mainly involving the left ventricle in mild cases, to massive necrosis with calcification involving both ventricular walls. The skeletal muscle changes consisted of hyaline degeneration, swollen cytoplasm, loss of striation, and sarcolemolysis, and some with hyperconcentrated muscle fibers. Areas of massive necrosis were heavily infiltrated with macrophages, satellite cells, some probably fibroblasts and few neutrophiles. Significant finding related to any possible infection was not noted. The results revealed an electropolation-related effect on these organs and tissues. A further investigation is worthy to follow.
P13 Ischemic Brain Damage after Ketamine and Xylazine Treatment in a Young Laboratory Monkey (Macaca Fascicularis)
1 Fujisawa Pharmaceutical Co., Ltd., Osaka, Japan and Although both ketamine and xylazine are frequently used for general anesthesia, the incidence of accidental problems associated with their usage is comparatively low in primates, similar to that for humans. Though these drugs are commonly used in combination in veterinary medicine, irreversible neuropathy has rarely been documented. We describe neural damage in a cynomolgus monkey after injection of ketamine and subsequent xylazine treatment. After the intramuscular injection of 20 mg/kg ketamine followed by 2 mg/kg xylazine, a 4-year-old female laboratory cynomolgus monkey manifested neurological abnormalities, including tetanic spasm with opisthotonus, nystagmus, and symptomatic epilepsia. An analysis of blood chemistry revealed severe elevation of levels of creatine phosphokinase. Histopathological deviations included acute neuronal necrosis and/or glial reaction in the cerebral cortex, nucleus lentiformis, hippocampus, cerebellar cortex and nucleus, and medulla oblongata; severe myocardial hemorrhagic necrosis; and hepatic subcapsular hematoma. This neural damage might be attributable to an ischemic condition in the brain due to systemic hemorrhage and subsequent decreased blood pressure following ketamine and/or xylazine treatment. Since both drugs have often been used as general anesthetics in veterinary medicine, attention should be paid to this rare case with neural damage.
P14 Moderate Diet Restriction Protects Against Doxorubicin-Induced Cardiotoxicity and LethalityUniversity of Louisiana at Monroe, Monroe, LA, United States Clinical use of doxorubicin (Adriamycin®), a broad-spectrum antineoplastic agent, is limited by its cardio- and nephrotoxic effects, mediated by oxygen radicals. Moderate diet restriction (DR) has a plethora of beneficial health effects. Effects of DR on drug-induced cardiotoxicity have been sparsely investigated. We hypothesized that moderate DR decreases oxidative stress, thereby protecting against doxorubicin toxicity. The objective was to investigate the effects of moderate DR against doxorubicin-induced cardio- and nephrotoxicity. Male Sprague-Dawley rats (250–275 g) were maintained on DR [35% of ad libitum (AL) fed rats, 42 days] followed by challenge with a LD100 dose of doxorubicin (DOX, 12 mg/kg, ip) on day 43. While all AL rats died between 7 to 13 days all the DR rats survived following DOX administration. To investigate the mechanism of this protection, biomarkers of cardiotoxicity and nephrotoxicity were measured over a time course of 12 days after DOX administration. Estimation of serum creatine kinase-MB, lactate dehydrogenase, and aspartate transaminase revealed high cardiotoxicity, exhibiting a biphasic response with peaks on 2nd and 12th day in the AL fed rats, while these biomarkers were not elevated in the DR rats. Nephrotoxicity, assessed by blood urea nitrogen, was elevated ~4-fold in the AL rats from day 4 until 12. In contrast, DR rats exhibited 55% lower renal dysfunction and fully recovered by day 10. These results indicate that DR rats are highly resilient to cardiotoxic and lethal effects of DOX. These findings illustrate the powerful beneficial cardiovascular effects of moderate DR as a life style option.
P15 Serum Sphingosine-1-Phospate and Sphinganine-1-Phosphate Are Increased in Horses with Fumonisin B1 Induced Vasogenic Injury
1 University of Illinois at Urbana-Champaign, Urbana, IL, United States and
The fumonisin mycotoxins alter sphingolipid biosynthesis resulting in increased concentrations of sphinganine (Sa) and, to a lesser extent, sphingosine (So), in serum, urine and tissues. Alterations in Sa and So are implicated in the mechanism of fumonisin toxicity and are used as biomarkers of exposure. So-1-phosphate (So-1-P) and, to a lesser extent, So are believed to play important roles in regulating vascular tone. In horses, fumonisin B1 induces neurotoxicity (leukoencephalomalacia), hepatotoxicity and cardiotoxicity. Because we suspect sphingolipid mediated cardiovascular dysfunction plays an important role in fumonisin induced toxicity, we characterized the dose-response relationship of fumonisin B1 induced changes in serum So, Sa and So-1-P and Sa-1-P using HPLC and LCMS (normalized to C20). Fumonisin B1 was administered IV at 0.20, 0.10, 0.05, 0.01 or 0 mg/kg BW/day (n = 3 or 4). Serum was obtained when horses were euthanized on day 28 or when neurologic signs became severe (
P16 Spontaneous Lesions of the Cardiovascular System in Purpose-Bred Laboratory Nonhuman PrimatesInveresk Research, Edinburgh, United Kingdom A retrospective study was carried out to determine the range, occurrence and incidence of spontaneously arising histopathological findings of the cardiovascular system in purpose-bred laboratory nonhuman primates. A total of 2427 animals, which included 2013 Cynomolgus monkeys (Macaca fascicularis), 308 common marmosets (Callithrix jacchus) and 106 rhesus monkeys (Macaca mulatta) from a total of 81 toxicological studies evaluated over a period of 5 years were analyzed. Only studies with equal number of male and female animals and a full range of routine histology tissues were included. An attempt was also made to standardize the range of pathological terms used by different pathologists. The most common findings in the heart included; inflammatory cell infiltrations of the myocardium (270), focal myocarditis (89), pericarditis (21), endocarditis (20) and congenital cysts (7). Myocarditis was more common in male (60%) than female animals. Perivascular inflammatory cell infiltration in the kidney, lung, brain, meninges, nerves and other tissues (150), localized vasculitis (35) and mineralization of vascular walls (17) were the most common findings involving blood vessels. Vascular mineralization and extramedullary haemopoiesis around blood vessels or the epicardium were more common in marmosets than other species. The findings of the study demonstrate the range and incidences of spontaneously occurring lesions of the cardiovascular system in purpose-bred laboratory nonhuman primates and suggest that care should be taken when interpreting such findings in toxicological studies.
P17 Studies to Identify Gene Changes in the Heart During Recovery from Heart Toxicity Induced by Bis(2-Chloroethoxy)Methane
1 National Institute of Environmental Health Sciences, Research Triangle Park, NC, United States Cardiotoxicity was induced in F344/N rats after dermal administration of bis(2-chloroethoxy)methane (CEM) (Dunnick et al. 2004 a,b). CEM heart damage occured by day 2 in all three regions of the heart (atrium, ventricle, interventricular septum) and was characterized by myofiber vacuolation, necrosis, mononuclear-cell infiltration, and atrial thrombosis. Ultrastructural analysis revealed that the primary site of damage was the mitochondrion, with disrupted cristae and loss of membrane structure, and that distention of the myocardial sarcoplasmic reticulum occurred. By day 5, even though dosing continued, the toxic lesions in the heart began to resolve, and by study-day 16 the heart appeared normal. Microarray gene analysis was used to test the hypothesis that gene changes at onset of toxicity (day 2) would differ from gene changes during recovery (day 5). RNA was extracted from the whole hearts of male rats at day 2 and day 5, and analyzed using Agilent rat oligonucleotide microarray. The significant gene changes (vs. control) at day 2 included 240 upregulated genes and 101 down-regulated genes. The significant gene changes at day 5 included 113 upregulated genes and 102 downregulated genes. Gene changes at day 2 were different from those at day 5, with the exception of 27 downregulated genes that were common on days 2 and 5. We hypothesized that specific gene changes enable the heart to recover from chemical-induced toxicity.
P18 The Evaluation of Cardiac and Other Soft Tissue Abnormalities in Rat Teratology Studies Using Magnetic Resonance Imaging (MRI)
1 National Institute of Environmental Health Sciences, Research Triangle Park, NC, United States and Magnetic resonance imaging (MRI) offers a novel, non-invasive method to evaluate rat fetuses in teratology studies. Pregnant Sprague-Dawley rats were orally treated on gestation day 11 with 120 mg/kg all-trans-retinoid acid (TRA) or 120 mg/kg 13-cis-retinoic acid (13CRA) suspended in corn oil. The treatment volume was 5 ml/kg body weight. Fetuses were collected on gestation day 20 by caesarean section. A rapid microwave fixation method was used with a 1:20 concentration of a gadolinium MR contrast agent in Bouin’s fixative in a conventional microwave oven for approximately 30 minutes. Two-dimensional, 0.5 mm thick multi-slice MR images were obtained in 20 minutes for both dorso-ventral and sagittal views of each fetus. Initial assessment of the MR images found that, compared to the body size of the fetus, the hearts of the treated fetuses were visibly larger than hearts of control fetuses. For further evaluation, hearts were segmented from the MR images and the volumes of the hearts were calculated. The average of heart volumes normalized by fetal body weight for control, TRA and 13 CRA fetuses are 0.0249, 0.0256 and 0.0255 ml/g BW respectively. This method allows for the screening of soft tissue malformations, like the cardiac abnormality found in this study. The non-invasive nature of MR allows for conventional techniques to subsequently be employed on fetuses after imaging. The combination of microwave fixation and MR imaging provides a quick, non-invasive method to evaluate fetuses in teratology studies.
P19 Thrombocytopenic Purpura in Göttingen MinipigsHuntingdon Life Sciences, East Millstone, NJ, United States Introduction: Thrombocytopenic purpura (TP) syndrome has been reported to occur spontaneously in sexually mature Göttingen minipigs at a low incidence (0.1%, 11/11637) in a closed breeding colony [Carasco et al, 2002]. TP is of unknown etiology and is also seen in normal pig. The corresponding syndrome in humans is von Willebrand’s disease. Results: In our laboratory, TP was noted in 2 Göttingen minipigs (5 month-old females) out of 306 tested between 2000 and 2003. A high incidence (0.65%) relative to that reported in the literature. Clinical signs: rapid onset and deterioration, pallor, lethargy, coalescing petechiae on the abdomen, face, neck and limbs, palpable swelling under the jaw and blood beneath the cage. Clinical pathology: marked thrombocytopenia, anemia, hematuria, proteinemia and glycosuria. APTT and PT were within normal range. Histopathology: interstitial hemorrhages were present in most organs; most lymph nodes contained large amounts of free erythrocytes in the subcapsular and medullary sinuses; the bone marrow was hypercellular and had numerous degenerating megakaryocytes with granulocytic hypereosinophilic cytoplasm and pyknotic nuclei; and the kidneys had minimal focal membranous glomerulonephritis. Discussion: The minipig is a non-rodent laboratory animal used extensively in safety evaluation of dermally administered materials and is increasingly being used as an alternative to dogs and non-human primates for evaluation of drugs administered by a variety of routes. The Göttingen minipig is popular for safety evaluation because of its small size, slow growth rate and non-pigmented skin. Therefore, as use of this animal increases, it is likely that an increasing number of cases of thrombocytopenic purpura will be observed.
P20 Toxicologic Pathology of Glutamate Receptors (GluRs)—An Opportunity for Pharmaceutical Development. Part I Human Heart*
1 Health Canada, Ottawa, Canada and Introduction: We demonstrated the localization of GluRs by immunohistochemistry in hearts of rats and monkeys. These receptors were observed within the conducting system, nerve terminals and in intramural ganglia. Here, we report on the observation of GluRs in human heart. Methods: Unstained histological preparations of cardiac samples were obtained from the Ottawa Hospital. Immunohistochemistry and pre-made Northern blots were used to localize the GluRs to specific anatomical regions of the heart. Results: Immunohistochemistry revealed differential expression for each subtype of GluR tested. GluRs were localized in the myocardium, the wall of blood vessels, conducting system, intramural nerves and ganglia. Results from the pre-made Northern blot showed the strongest signal in the right ventricles for the receptors subtypes NMDAR1 and KA2. The expression of NMDAR 1 is strong in all areas, absent in the aorta. Discussion: We demonstrate that GluRs are present in human heart. The cellular specific distributions of these receptors support the view that GluRs may play a role in cardiac function and in the mediation of cardiotoxicity. Further functional and pharmacological characterization of these GluRs in the heart could lead to the development of therapeutic agents. *This study was supported by a grant from the Office of the Chief Scientist and approved by the Health Canada Research Ethics Board
P21 Ultrasonic Analysis, a Tool for Early Detection of Cardiotoxic Lesions
1 National Institute of Environmental Health Sciences, Research Triangle Park, NC, United States and Exposure to environmental toxicants can contribute to the initiation of heart disease, of which early diagnosis can be vital. In this study, histopathological and ultrasonic examinations of heart disease induced in the mouse bis(2-chloroethoxy)methane (CEM) model were compared. Nine 5-week-old female B6C3F1 mice were exposed dermally to 0, 400, or 600 mg/kg CEM for 3 consecutive days. Ultrasonic analysis was conducted using the Visualsonics Vevo 660 with a 30 MHz probe prior to treatment and approximately 2 hours after the third exposure. During both imaging sessions, mice were anesthetized with isoflurane. While histopathological analysis showed no evidence of treatment-related lesions, an M-mode echocardiogram across the short axis of the heart revealed thinning and abnormal structure of the interventricular septum and ventricular wall, an abnormal pattern of heart rhythm, and shallow systolic and diastolic beats. This investigation demonstrates that early functional cardiotoxic alterations not detectable histopathologically can be revealed by ultrasound, an important tool in our armamentarium used to discover environmental initiators of heart disease.
P22 Vascular Injury Induced by Sphingosine-1-Phosphate Receptor Agonists in DogsMerck Research Laboratories, West Point, PA, United States Sphingosine-1-phosphate (S1P) agonists have been proposed for therapeutic use in prevention of organ transplant rejection. As part of the preclinical evaluation, S1P agonists designated Compounds A, B, C, and D were evaluated in 2 week oral toxicity studies in beagle dogs. These S1P agonists were evaluated for binding to S1P receptors 1, 2, 3, 4, and 5 with Compound D having high specificity for the S1P-1 receptor. The primary dose limiting toxicity for all 4 compounds was degeneration of intramural arteries in the left ventricle of the heart. The changes in the small arteries were characterized by degeneration or necrosis of individual smooth muscle cells of the tunica media with associated collections of erythrocytes and/or cellular debris and edema within the adventitia. Mitosis was observed in the tunica media and adventitia indicating repair. The lack of a cellular inflammatory response was attributed to the pharmacologic properties of S1P agonists and their ability to rapidly and dramatically reduce the population of circulating lymphocytes and neutrophils in dogs. Compounds B and C were evaluated in a cardiovascular safety pharmacology study in telemeterized dogs and each elicited a decreased heart rate and increased blood pressure. Degeneration of intramural arteries in the heart occurred in dogs after 2 weeks of oral dosing with S1P agonists at relatively low doses.
P23 Diabesity: A Polygenic Model of Diet-Induced Obesity (DIO) in Crl: CD (SD) Rats
1 Merck Research Laboratories, West Point, PA, United States The Ad Libitum (AL), overfed, outbred CD rat is a model for DIO in humans. Obesity is among the top 10 human health risks leading to type 2 diabetes, hyperlipidemia, heart disease, hypertension, stroke, renal disease, osteoarthritis and cancer. While monogenetic mutations in rats (Zucker or JCR rats) model human obesity and the metabolic syndrome, these null mutations in specific genes do not represent the polygenic human syndrome of obesity-driven type 2 diabetes or diabesity. AL overfed outbred CD rats develop a DIO that progresses to diabesity, with 40% body fat, hyperlipidemia, hyperinsulinemia, endocrine dysfunctions, nephropathy and cardiomyopathy. They also develop early reproductive senescence and tumors in pituitary, pancreatic islets, adrenals and other tissues. This diabesity syndrome in AL overfed, DIO CD rats is readily accelerated by changing diets or controlled by dietary restriction (DR). Feeding of balanced diets at 75, 70 or 50% of AL food intake show, in a "caloric dose dependent" fashion, the morphological and metabolic differences between the DIO and DR CD rats, and demonstrates temporal changes in the expression of phenotype that models the polygenic human disease syndrome. At moderate levels of DR (70–75% AL) a milder DIO phenotype (20–25% body fat) develops that delays the onset of morbidity and mortality associated with the DIO syndrome. AL-overfeeding of CD rats provides a needed and untapped model of gradual onset DIO and adult onset diabesity that needs further profiling of the temporal patterns of polygenic gene expression and key molecular targets for potential therapy. Also, moderate DR controls the DIO syndrome and improves the CD rat as a DIO toxicology model that can distinguish the onset of DIO and background aging from true drug treatment effects and toxicological endpoints.
P24 Investigation of Apoptosis in the Murine Olfactory Epithelium Evoked by Vincristine Sulphate in Comparison with that Induced by Unilateral BulbectomyDaiichi Pharmaceutical Co., Ltd., Tokyo, Japan In order to investigate an initial event of olfactory epithelial apoptosis, vincristine sulphate (VCR) was intravenously administered once at 1.17 and 1.95 mg/kg to male BALB/c mice on Day 1. The olfactory epithelium, nerve and/or bulb were sequentially examined by light- and electron-microscopy 6 and 24 h (Day 2) later, and on Days 4 and 15. Further, BrdU-morphometry of the olfactory epithelium was performed at 6 and 24 h post-dose, and whole-body radioluminography was conducted 1 and 24 h later. Unilateral bulbectomy (UBT) group was set to compare neurectomy- and VCR-induced early changes in the olfactory epithelium. Apoptosis and increased number of mitosis with a tendency for a decrease in BrdU-positive cell counts were observed in olfactory epithelial cells at 6 h post-dose. Apoptosis and decreased cell counts became more pronounced 24 h later, but disappeared on Days 4 or 15. On Days 4 and 15, minimal axonal degeneration was seen in the olfactory nerve, but not in the olfactory bulb. Semiquantitative drug levels of VCR in the ethmoturbinals were approximately 2 times of those in blood at either 1 or 24 h post-dose. In contrast, UBT showed no effect on mitosis and BrdU-positive cell counts at 6 h post-dose, and also severe lesions in the olfactory epithelium and nerve on Days 2, 4 and/or 15. These results suggest that the initial event of apoptosis in murine olfactory epithelial cells induced by VCR would be mitotic arrest with high drug distributions, unlike that induced by UBT.
P25 The Transduction of Rat Submandibular Glands by an Adenoviral Vector Carrying the Human Growth Hormone Gene is Associated with Limited and Reversible Changes at the Injection Site
1 Integrated Laboratory Systems, Research Triangle Park, NC, United States Adenoviral vectors have been investigated for use in gene therapy to correct for single protein deficiency diseases via the expression of transgenes. The purpose of this study was to evaluate the response of F344 rats to intraductal infusion of the right submandibular salivary gland with an adenoviral vector encoding the gene for human growth hormone (Ad-hGH), accompanied by co-administration of hydroxychloroquine (HCQ). Male and female F344 rats were sacrificed at 3 and 28 days post injection and all tissues were evaluated. The six test groups were: vehicle saline, vehicle HCQ, 6 x 109 particles of Ad-hGH in saline, 1.5 x 1011 particles of Ad-hGH in HCQ, 6 x 109 particles of Ad-hGH in HCQ, and 2.4 x 108 particles of Ad-hGH in HCQ. There were 4 animals in each 3-day sacrifice group and 8 animals in each 28-day sacrifice group. The right submandibular salivary gland (injection site) was the primary target organ with microscopic lesions characteristic of a moderate insult. At 3 days post injection, all rats, with the exception of the saline control groups, had variable degrees of glandular acute inflammation, degeneration and necrosis, with more severe lesions in the higher dose groups. At 28 days post injection, these rats had milder inflammation and degeneration with variable degrees of regeneration. In conclusion, the effects on the salivary glands are predominantly reversible as indicated by the milder inflammation and degeneration in the day 28 rats concomitant with mild to moderate acinar regeneration. Therefore, the vector appears relatively innocuous with very limited tissue toxicity. This work was supported by National Institute of Environmental Health Sciences [E01-ES-95446], NIEHS [N01-ES-75408] and NIDCR [ZO1 DE000336-23].
P26 Infusion of Cynomolgus Monkeys with a Novel Conditionally-Replicating Oncolytic Adenovirus: Pathologic Profile
1 Drug Safety and Metabolism, SPRI, Lafayette, United States A conditionally-replicating oncolytic adenoviral vector (01/PEME) was engineered to confer attenuated replication in normal human cells and replicate preferentially in cells with dysregulated p53 and Rb/E2F pathways. The safety of 01/PEME was assessed in female cynomolgus monkeys (2/group) following the infusion of 1 x 1011, 5 x 1011, 1 x 1012, or 5 x 1012 viral particles/kg (p/kg). Early morbidity/mortality occurred in both high-dose monkeys. Both monkeys exhibited prolonged coagulation time, thrombocytopenia and elevated serum transaminase. Necropsy findings included liver friability, pale discoloration, and accentuated lobular pattern. Multifocal hemorrhages were present in the gastrointestinal tract, skin, eyelids, and lungs. Histopathologic findings included diffuse hepatic necrosis, inflammation, and intranuclear inclusions. Vascular inflammation with few thrombi and rare endothelial cell intranuclear inclusions were present in the lungs of one high-dose monkey. Vascular inflammation was present in the eyes or gallbladder from monkeys given 5 x 1011 or 1 x 1012 p/kg. Ultrastructural evaluation of liver from one high-dose monkey revealed amorphous to granular intranuclear inclusions but no morphologic evidence of complete adenoviral replication such as viral particles or [para]crystalline arrays. Adenoviral hexon mRNA was detected in the liver from high-dose monkeys suggesting that abortive replication had occurred. Findings in this study were consistent with reported findings following the administration of empty adenoviral capsids, non-replicating adenoviral vectors, and conditionally-replicating adenoviral vectors, which can lead to acute liver failure and death. Our findings also suggest that normal monkey cells may allow partial/abortive replication of 01/PEME.
P27 Combinatorial Rat Panels for Predictive Toxicology
1 PhysioGenix, Milwaukee, WI, United States and In drug development today, over 90% of drugs fail in clinical trials. The problem is in part due to the lack of genetic diversity in animal models used during the testing phase of preclinical trials. To address this, a unique animal model system that predicts genetic components has been developed. These unique panels are comprised of six F1 hybrid strains combinatorially bred from four inbred parental strains. This combinatorial breeding enables the same genome to be tested in different backgrounds in a controlled fashion. These panels capture over 80% of the genetic diversity found in the rat genome and enables drug responses to different genome backgrounds to be tested simultaneously. The panels emulate the genetic diversity and heterogeneous genome background found within the human population for easier detection of drug toxicity. Previous studies with a nephrotoxin, gentamicin, and a hepatotoxin, methapyrilene, and current studies with hepatotoxins acetaminophen and tacrine reveal hybrid strains that were sensitive and resistant to the toxicity. These results emphasize the necessity of testing across a variety of genome backgrounds to avoid future drug failures in clinical trials. Researchers who discover a differential toxic response among these strains will use this information as a starting point for drug optimization, drug rescue, and mechanistic studies to discover genetic basis of toxicity.
P28 Popliteal Lymph Node Examination in Immunotoxicity Assessment in the Rat
1 Pfizer Global Research and Development, Groton, CT, United States and Although examination of peripheral lymph nodes is part of routine safety assessment, the reliability and validity of lymph node weights and histology has not been critically assessed. This study seeks to compare the sensitivity of weights and histology of spleen, thymus and popliteal lymph nodes (PLN) in detection of immunotoxicity caused by cyclophosphamide. Male Sprague-Dawley rats (n = 10/group) were administered cyclophosphamide at 2, 7 and 12 mg/kg for 10 days. Brain, left and right PLNs, spleen and thymus were weighed and processed for histology. Organ-to-brain weight ratios were calculated, and histologic sections were examined by one pathologist blinded to treatment. Within-animal left and right PLN weights were significantly but weakly correlated (p = 0.0005, r2 = 0.26). Cyclophosphamide caused dose-dependent decreases in all lymphoid tissue weights, of similar magnitude at the low and mid doses, but of greater magnitude in thymus at the high dose. Paired PLN weights showed greater within-group variability than spleen at all doses, and greater variability than thymus in control and low dose groups. Histologic changes due to treatment were noted at the low dose in the spleen (1/10) and PLN (1/10), at the mid dose in thymus (4/10), PLN (6/10) and spleen (8/10), and in all tissues in all high dose rats. The results suggest that PLN weights are more variable than spleen and thymus weights in rats, and weighing PLNs does not increase sensitivity of routine screening. If PLNs weights are needed, both sides should be weighed because of size variability. Histology of PLNs may be useful in detection of immunotoxicity, but does not appear to increase overall sensitivity.
P29 Toxicologic Pathology of Glutamate Receptors (GluRs)—An Opportunity for Pharmaceutical Development. Part II: Inflammation and Lymphoid Organs
1 Health Canada, Ottawa, Canada and Introduction: Evidence suggests that the GluRs have a role in neuroimmunomodulation and fast acting signalling pathways between the neural and immune systems. To assess the potential relevance to human, we investigated the presence of GluRs in humans and monkeys. Here, we report on the observations in a human heart with sarcoidosis, tissue arrays, and tissues from monkeys with chronic inflammation. Methods: Unstained histological preparations of heart were obtained from the Ottawa Hospital. Tissues from monkey uterus were obtained from Health Canada archive. Immunohistochemistry and pre-made Northern blots were used to analyse the different subtype of GluRs. Results: In the heart with sarcoidosis, GluRs were differentially expressed within the foci of inflammation, and the granulomas seen within the conducting system. Giant cells had affinity for GluR 2/3, mGluR 2/3 and NMDAR 1. The monkey uterus showed NMDAR 1 immunolabeling with specific affinity for chronic inflammatory infiltrates. Mast cells show strong affinity for the anti-NMDAR 1. Tissue arrays show that NMDAR 1 had the strongest expression. Discussion: The expression of GluRs in lymphoid tissues of human, monkey and rodents suggest that these receptors may be involved in immune functions. Expression within areas of inflammation suggests that they may be involved on the pathobiology of inflammation. Further characterization of these GluRs in these tissues could lead to the development of therapeutic agents. Supported by a grant from the Office of the Chief Scientist and approved by the HC Canada Research Ethics Board.
P30 Histopathology in Acute Poisonings with Alcohol Beverages Seized from Market
1 Pavlov State Medical University, St. Petersburg, Russia and Introduction: The purpose of the study has been to assess toxicity of strong alcoholic beverages illegally sold in Russia and to compare the toxic metric data with histopathology. The following methods were used: screening of 38 samples of alcoholic beverages on spermatozoa motility, toxic metric parameters assessment, behavior and pathology of internal organs evaluated with hematoxylin-eosin staining, PAS-reaction, sudan III and IV staining in male albino-rats and mice. Results: Some alcohol beverages had relatively higher toxicity when compared with standard ethanol solutions. The behavioral tests found no differences between the groups. Standard ethanol solution given orally to rodents (1 LD50) resulted in brain edema, heart plethora, mild alveolar edema, acute venous plethora and mild steatosis in the liver and in the kidney, mild dysproteinosis in the kidney. Several samples of alcoholic beverages resulted in severe dystrophic changed in brain, heart, lungs, liver and kidneys. Conclusion: Acute exposure to some alcoholic drinks causes severe liver and kidney steatosis in rodents, whereas acute poisoning with ethanol solution doesn’t result in these disorders. It is necessary to note that the tested samples being evaluated with mass spectrometry contained no toxic admixtures (except cognac samples). The non-toxic flavorings and additives to alcoholic beverages are supposed to modify ethanol toxicity.
P31 Pathology and Behavioral Changes in Psilocybine-Containing Mushrooms Poisonings in Rodents
1 Pavlov State Medical University, St. Petersburg, Russia and Introduction: There is high incidence of Psilocybine-containing mushrooms (PCM) abuse in St. Petersburg region. PCM poisonings manifest both with mental and internal disorders such as tachicardia, vomiting, hyperthermia and kidney disturbances etc.
The aim of the present study was to evaluate kidney, liver and brain pathology in poisonings caused by PC Methods: Screening of the effective dose of PCM was performed on male albino-rats (n = 96). Behavioral tests selected as an endpoint for screening included aversive stimulation, passive avoidance conditioning and open field. 45 male albino-rats received 1.5 g/kg of PCM suspension once a day for 3 days or normal saline via gastric tube. Kidney, liver and brain pathology were evaluated with basic histological techniques and histochemistry after the euthanasia. Results: 0.75 g/kg was found as an effective dose that accelerates aggressive response in rats. Open field assessment and passive avoidance conditioning didn’t provide statistical difference between groups. Dysproteinosis and steatosis were the most common signs of liver and kidney damage. The last one reveals also mesangial cell number increase. It was found that succinate dehydrogenase, alkaline phosphatase, 5-nucleotidase and NADH2dehydrogence activities decrease in the kidney, and lactat dehydrogenase activity increases. Monoamineoxidase activity decreased in the brain and cholinestherase increased, representing behavior disorders. Conclusion: Kidney, liver and brain disorders related to PCM may be related to the disturbances in oxidative phosphorylation due to PCM poisoning.
P32 Increases in Serum Transaminases Related to Ketamine Anesthesia in Chimpanzees
1 Biogen Idec Inc., Cambridge, MA, United States and CBE11, a humanized monoclonal antibody to lymphotoxin beta receptor (LTBR) currently in Phase 1 clinical trials for solid tumor indications, cross-reacts specifically with only chimpanzee and human LTBR. The chimpanzee has numerous disadvantages as a preclinical model because of the limited safety data that can be obtained and because they must be fasted and anesthetized prior to any treatments. In a repeat dose escalation study with CBE11 in chimpanzees, the animals were anesthetized with 10 mg/kg ketamine prior to blood collection, physical exams, and administration of the vehicle or test article. Increases in serum alanine transaminase (ALT) and aspartate transaminase (AST) above historical data ranges were observed in vehicle and test article exposed animals three days post-dosing. Enzyme elevations were more pronounced post-dosing than elevations that occurred following anesthesia for purposes not including dosing (blood collections, physical exams). Further analysis demonstrated that on days of dosing, animals were anesthetized for up to 2 hours longer and given 3–4 fold greater cumulative doses of ketamine than on days of study when routine blood draws were performed. While generally not regarded as hepatotoxic, increases in ALT and AST have been observed in rats, rabbits and humans anesthetized with ketamine, supporting a potential role for ketamine as a hepatotoxicant. The use of a vehicle control in all preclinical studies is essential to distinguish test article related effects from confounding factors. The use of ketamine in preclinical studies evaluating the safety of therapeutics may confound the interpretation of changes in indices of hepatocellular damage.
P33 Neurotoxic Effects of Zoniporide: A Selective Inhibitor of the Sodium-Hydrogen Exchanger Isoform 1 (NHE-1)
1 Pfizer Global Research & Development, Groton, CT, United States Zoniporide is a selective inhibitor of the Na+-H+ exchanger-1 intended for the reduction of myocardial ischemic injury. It was administered by continuous intravenous infusion in preclinical studies (up to 1-month) to support its use in clinical trials (up to 7 days). Some treated human subjects experienced reversible peri-ocular pain and mild, transient paresthesia, although 2-week pre-clinical studies showed no effects on nervous tissues. However, a 1-month rat study (0, 10, 30, 150 mg/kg) showed minimal nerve fiber (axonal) degeneration (spinal cord and/or sciatic nerve). Follow up 1-month rat neurotoxicity (0, 150 mg/kg) and dog (0, 2, 10, 20 mg/kg) studies included comprehensive sampling and evaluation of nervous tissues, periodic functional observational batteries and electrophysiology assessments (rat), and weekly neurological examinations (dog). Rat findings included slowing of caudal nerve conduction velocity (NCV) (days 14 and 28), nerve fiber degeneration in spinal cord (dorsal funiculus), dorsal roots and ganglia, and radial, sciatic and tibial nerves. In dogs, the patella reflex was a decreased or absent (day 7), minimal to marked nerve fiber degeneration (dorsal root ganglia) and minimal nerve fiber degeneration (spinal dorsal roots and funiculi) were observed. The preclinical findings were consistent with a specific effect on sensory nerve fibers. Since histological changes in nervous tissues were not observed in 2-week studies, the patella reflex effect and decreased NCV reflect changes in nerve function that preceded morphological evidence of nerve damage. These results demonstrated that zoniporide produces a peripheral sensory neuropathy and establishes the importance of careful preclinical evaluation of neurological function.
P34 Nasal Effects in Rats Associated with Oral Gavage DosingSanofi-Aventis, Drug Safety Evaluation, Bridgewater, NJ, United States
Inflammation and epithelial degeneration/necrosis in the nasal mucosa of rats were identified in a 7-day oral gavage toxicity study at test article doses
P35 Scanning Raman Microprobe Analysis as a Tool in Toxicologic Pathology
1 Sanofi-Aventis, Drug Safety Evaluation, Bridgewater, NJ, United States and
AVEXXX is a highly insoluble interleukin-4 expression inhibitor that was in development for the treatment of asthma. As part of a preclinical safety program, a 13-week rat inhalation toxicity study was conducted using a dry powder formulation of micronized, crystalline AVEXXX, blended in lactose. Rats were exposed to the powder formulation of AVEXXX at 0, 0.5, 5 or 50 mg/kg/day for four hours/day for 13 weeks using "nose-only" inhalation exposure chambers. At necropsy, pale foci were observed in the lungs at 5 and 50 mg/kg/day. Microscopic examination of the lungs revealed accumulations of macrophages containing phagocytized, birefringent crystalline material (interpreted as presumptive AVEXXX) in the alveolar airspaces of rats at
P36 Obstructive Protein Cast Nephropathy in Monkeys Treated with Molecules Containing a Carboxylic Acid MoietyPfizer Global Research and Development, Ann Arbor, MI, United States The carboxylic acid moiety is an important component of major drug classes such as the nonsteroidal anti-inflammatory drugs (NSAIDS) and anticonvulsants. The carboxylic acid moiety undergoes extensive glucuronidation to increase water solubility and renal elimination. We have observed a renal toxicity consistent with an obstructive nephropathy in cynomolgus macaques treated with different small molecule drugs having a common carboxylic acid moiety. Renal findings are typically characterized by cast formation, interstitial inflammation, tubular dilatation and mild expansion of the medullary interstitium. Under electron microscopy (EM) cast material consists of filamentous material, identified as Tamm-Horsfall protein (THP) by immunohistochemistry. Crystalloid material has been identified in membrane bound vacuoles within tubular epithelial cells by EM. It is hypothesized that formation of micro-crystals in the urinary tubular spaces induces aggregation of THP protein leading to cast formation. Alterations in renal function tests (serum BUN and creatinine) have been dependent on the severity of morphologic alterations. It is plausible that carboxylic acid containing compounds, when excreted primarily by the kidneys, can alter the ionic concentration and pH within the urinary micro-environment of the nephron. Such physico-chemical alterations can facilitate the formation of crystals and THP casts. Although many marketed drugs such as ibuprofen, naproxen, diclofenac and even aspirin, contain a carboxylic acid moiety, clinical cases of drug induced obstructive nephropathy that mimic the pathology observed by us in monkeys, have not been documented in humans. Therefore, the relevance of these findings in the context of human risk assessment may be questionable.
P37 Histopathological Evaluation of Dog Testes: The PitfallsHuntingdon Life Sciences, East Millstone, NJ, United States Identification of toxicologically induced changes in dog testes and epididymides is complicated by a number of problems relating to immaturity, spontaneous background pathology and small group sizes used in routine regulatory studies. These confounding factors can result in generation of false positive, false negative or uninterpretable results. This poster illustrates the major issues that the pathologist should be aware of when evaluating the male reproductive tissues of the dog. Dogs mature anywhere between 7–12 months of age. Regulatory guidelines recommend starting studies with dogs 5–6 months of age. Depending on the duration of the study, dogs coming to necropsy may be immature, peripubertal or fully mature. If dogs are immature testicular toxicity cannot be identified since there are no germ cells present. Dogs that are peripubertal cannot be distinguished from mature dogs undergoing spermatogenic degeneration. If a study has a mixture of mature and peripubertal dogs unevenly distributed through control and treated groups, this may be mistaken for a treatment related effect. Normal Beagle dogs have a high incidence of background degenerative changes in the testes including 30% hypospermatogenesis, 30% tubular hypoplasia/atrophy and almost 100% incidence of degenerating (multinucleated) germ cells. It is important to recognize the characteristics of these spontaneous lesions and have an accurate background database to interpret these changes. When small group sizes of only 3/group are routinely used, the ability to detect a treatment related effect, over and above such a high background incidence is very poor.
P38 An Approach to the Interpretation of Renal Tubule Proliferative Lesions in F344 Rats with Advanced Chronic Progressive Nephropathy (CPN)1 EPL, Research Triangle Park, NC, United States It is accepted that, in the rodent kidney, tubule profiles with a complex hyperplastic lining, "Atypical Hyperplasia" (ATH), represent a preneoplastic lesion in the biological continuum that may lead to adenoma and, subsequently, carcinoma development. Chronic Progressive Nephropathy (CPN) is a spontaneous disease of the rat kidney characterized by a spectrum of tubule, glomerular, and interstitial histologic lesions. Advanced CPN may contain many tubule profiles of an apparently proliferative nature that can be a delimma for diagnosis and interpretation. These tubule profiles may represent tangential sections; tubule cell hypertrophy; intratubular cell clusters; dilated altered tubules; cystic altered tubules; and solid tubule profiles. This presentation will describe these different tubule profiles comparing them with unequivocal foci of ATH, and incipient adenomas. Our recommendations are based on the examination of large numbers of kidneys with advanced CPN, characterizing these lesions with a marker of cell proliferation, and serial sectioning of selected examples for tracing tubule profile development. As a result, we have attempted to define criteria that we hope will assist in the interpretation of various tubule profiles encountered in CPN.
P39 Classification of Canine Urinary Bladder Urothelial Tumors Based on the World Health Organization/International Society of Urological Pathology Consensus Classification
1 MPI Research, Mattawan, United States Currently there is not a single widely accepted classification system for canine urinary bladder tumors. The accurate and consistent classification of these tumors in dogs is essential for comparative research, and a number of recent studies have used the dog as an effective comparative model of human invasive urinary bladder cancer, especially for testing the effectiveness of various chemotherapeutic agents. The purpose of this study was to determine if the recently described World Health Organization/International Society of Urological Pathology (WHO/ISUP) urothelial tumor consensus classification used in human medicine could be applied to canine tumors. One hundred canine urinary bladder urothelial (transitional cell) tumors, including roughly equal numbers of benign and malignant forms, were retrospectively categorized using the WHO/ISUP classification. The tumors were randomized, blinded, and classified by three veterinary pathologists and two MD pathologists. The human WHO/ISUP classification system was found readily applicable to the dog and the various diagnoses and grades assigned to the tumors were highly consistent between all reviewing pathologists.
P40 Comparative Uterine Cellular Expression and Distribution of c-fos, Estrogen Receptor Alpha and p38 AlphaPfizer Global R&D, Ann Arbor, MI, United States
Key Words: c-fos • estrogen receptor
c-fos is an immediate early proto-oncogene that interacts with Activator Protein-1 (AP-1) in the nucleus to initiate a cascade of gene induction events that leads to uterine cell proliferation. Binding of estrogen to its receptor (ER
P41 Cytoplasmic Overexpression of Thioredoxin in the Cholangiocarcinoma and Dysplastic Bile Ducts in the Cholangiocarcinoma Hamster Model
1 Department of Veterinary Medicine, Kangwon National University, Chuncheon, South Korea Thioredoxin (Trx) is a redox regulating system that regulates cell proliferation and death in eukaryotic cells. Its overexpression has been shown to be associated with the survival advantage of several malignant tumors including lung and colorectal cancers. However, the possible role of Trx in cholangiocarcinogenesis is not studied, yet. To determine the aberrant expression of Trx in cholangiocarcinoma (ChC), we investigated immunoreactivity of Trx in the 12 cases of ChC and surrounding hyperplastic biliary ducts obtained from hamster ChC model as well as 5 cases of normal bile ducts. Expression of Trx in the proliferating biliary ducts of the 10 cases of interim stage (8 weeks after liver-fluke infection) of hamster ChC model was also evaluated. Overexpression of Trx was typically found in the cytoplasm of ChC and dysplstic bile duct cells, but the well-organized hyperplastic bile ducts and normal bile ducts were negative to weak positive. Further, it was also of interest that the cancer cells invading the surrounding parenchyma were particularly strong positive for Trx. On the other hand, the large hepatic or common bile ducts severely dilated by presence of the infected liver-flukes also showed strong immunoreactivity for Trx, not only in cytoplasmic but also in the nuclear location. Those unique expression patterns of Trx in the neoplastic and non-neoplastic bile duct cell components strongly suggested that Trx, a putative oncogene, might play an important role in neoplastic changes of the candidate bile duct cells for cholangiocarcinogenesis in the hamster ChC model.
P42 Enhanced Reduction of Aflatoxin B1-Induced Preneoplastic Hepatic Nodules in Rats Fed a Combination of Phytochemicals from Cruciferous VegetablesUniversity of Illinois, Urbana-Champaign, IL, United States A short term in vivo carcinogenicity study using aflatoxin B1 (AFB1) was performed to evaluate chemoprotection by two bioactive cruciferous compounds, indole-3-carbinol (I3C) and crambene (CHB). I3C upregulates certain Phase I enzymes, and both induce certain glutathione S-transferases, which theoretically should protect against AFB1 carcinogenesis. To determine whether these compounds in the diet are protective individually or in combination, groups of male F344 rats were fed diets supplemented with these compounds. Groups included a negative control (no AFB1, CHB or I3C), a 0.15% CHB group, a 0.165% I3C group, a "high dose" combination group (0.15% CHB, 0.165% I3C,) a "low dose" group (0.030% CHB, 0.033% I3C), and a group fed neither. All but the first group received AFB1 after two weeks of dietary pretreatment. Liver sections were scored for lesions—karyomegaly, mitotic rate, apoptotic index, biliary hyperplasia,—and evaluated immunohistochemically for expression of the preneoplastic marker, glutathione S-transferase-Pi (GSTP). The I3C and CHB groups in general were moderately protected against AFB1 while the low dose group was not protective. The high dose group had scores close to the negative controls. For log transformed two and three dimensional GSTP data, the high dose group demonstrated synergistic reduction in GSTP-positive area and additive reduction in GSTP-positive volume compared to the reductions in area and volume in the CHB and I3C groups. The low dose had no effect at all. In conclusion, high combined dietary doses of I3C and CHB demonstrated enhanced protection from AFB1 carcinogenesis. Supported by AICR #00B016.
P43 Gingival Carcinogenicity in Female Harlan Sprague-Dawley Rats Following Two-Year Oral Treatment with 2,3,7,8-Tetrachlorodibenzo-p-dioxin and Dioxin-Like Compounds
1 National Institute of Environmental Health Sciences, Research Triangle Park, NC, United States We evaluated gingival toxicities induced by chronic exposure of female Harlan Sprague-Dawley rats to dioxin and dioxin-like compounds (DLCs) and compared them to similarly induced oral lesions reported in the literature. This investigation represents part of an ongoing initiative of the National Toxicology Program to determine the relative potency of chronic toxicity and carcinogenicity of polychlorinated dioxins, furans, and biphenyls. For 2 years, animals were administered by gavage 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; a binary mixture of PCB126 and 153; or a binary mixture of PCB126 and 2,3',4,4',5-pentachlorobiphenyl (PCB118); control animals received corn oil-acetone vehicle (99:1) alone. A full complement of tissues, including the palate with teeth, was examined microscopically. In the groups treated with TCDD and the mixtures of TCDD, PCB126, and PeCDF; PCB126 and 153; and PCB126 and 118, the incidences of gingival squamous hyperplasia increased significantly. In the groups treated with TCDD, PCB126, and the mixture of PCB126 and 153, squamous cell carcinoma (SCC) in the oral cavity increased significantly. This investigation constitutes the first report documenting that chronic administration of dioxin-like PCBs can induce gingival SCC in rats. These results indicate that dioxin and DLCs target the gingiva of the oral cavity, in particular, the junctional epithelium of molars.
P44 Mechanisms of Exocrine Pancreatic Toxicity Induced by Oral Treatment with 2,3,7,8-Tetrachlorodibenzo-p-dioxin in Female Harlan Sprague-Dawley Rats
1 National Institute of Environmental Health Sciences, Research Triangle Park, NC, United States In previous 2-year studies of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) conducted by the NTP using female Harlan Sprague-Dawley rats, acinar-cell vacuolation, atrophy, inflammation, and arteritis developed at high incidence, and a rare occurrence of pancreatic acinar-cell adenomas and carcinomas was noted. In this investigation, we sought to identify the mechanism involved in the early formative stages of acinar-cell lesions. Pancreas from animals treated for 14 and 31 weeks with 100 ng TCDD/kg body weight or corn oil vehicle was examined immunohistochemically and/or morphometrically. Acinar-cell kinetics were analyzed using staining with hematoxylin and eosin and proliferating cell nuclear antigen. Expressions of cytochrome P450 (CYP) 1A1 and aryl hydrocarbon receptor (AhR) were evaluated to assess direct effects of TCDD exposure. CCK-A receptor (CCKAR) and duodenal cholecystokinin 8 (CCK) revealed the associations of dioxin treatment with hormonal changes. Amylase localization showed acinar structural changes that could affect enzymatic production. Increased apoptotic activity in acinar cells occurred in 14- and 31-week-treated animals, with an increase in proliferative activity in the latter. Also in the latter, in the vacuolated acinar cells, CYP1A1 was overexpressed, while statistically significant decreases in expressions of AhR, CCKAR, and amylase occurred. The intensity of CCKAR expression increased in nonvacuolated acinar cells; a decrease in the size of CCK-positive epithelial cells occurred in duodenum. Increase in CYP1A1 and decrease in CCKAR expressions in vacuolated acinar cells may be involved in the pathogenesis of pancreatic lesions.
P45 Morphological and Immunohistochemical Characteristics of Rat Pineal Parenchymal Tumor Observed in National Toxicology Program StudiesNational Institute of Environmental Health Sciences, Research Triangle Park, NC, United States Since rat pineocytoma, or pineal parenchymal tumor (PPT), occurs infrequently, its morphological features have required elucidation. We studied 11 F344 and 4 SD rats [5 males (m), 10 females (f)] diagnosed with this tumor by the National Toxicology Program. We attempted to classify and compare it to the human type using criteria generated by the World Health Organization. Benign pineocytoma occurred in 4 animals; malignant pineocytoma occurred in the remainder. Pineocytomas showed compressive proliferation and often displayed a lobular or nest pattern forming pineocytomatous pseudorosettes resembling large Homer-Wright rosettes and lacking a central canal or capillary; Flexner-Wintersteiner-like (FW) rosettes with central canals and occasional mitoses were also noted. Malignant tumors exhibited invasive proliferation into adjacent brain parenchyma, hemorrhagic areas, necrotic foci, more mitoses, and sporadically occurring pineocytomatous and FW rosettes; although 3 cases showed some migration into the third or fourth ventricle, no extracranial or cerebrospinal-fluid metastasis occurred. Based on human criteria, these were diagnosed as pineocytomas (3m, 2f), pineal parenchymal tumor of intermediate differentiations (5f), and pineoblastomas (2m, 2f); from these classifications, respectively, 4 pineocytomas, 5 pineal parenchymal tumor of intermediate differentiations, and 2 pineoblastomas displayed malignancy with invasion into surrounding tissues. Both benign and malignant tumor cells showed strong immunopositivity for synaptophysin and neuron-specific enolase, markers indicating pineal origin. Although PPT in rats was similar histologically to that in humans, biological behaviors were not identical. In the rat, more invasion into surrounding tissues occurred, and female predominance was evident.
P46 PPAR
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-induced So increase in HepG2 hepatocytes, suggesting an interaction between the JNK signaling pathway and the sphingolipid metabolic pathway. To determine whether the JNK pathway is also involved in FB1-induced sphingolipid alteration, we examined the effect of SP600125 (10 µM) on Sa and So in A10 aortic smooth muscle cells, H9C2(2-1) cardiomyocytes and HepG2 hepatocytes after 24, 48 and 72 hr treatment with FB1 (10 µM). FB1 caused a marked and sustained increase in Sa in all 3 cell types, and a slight transient increase in So in HepG2 cells at 24 hr or slight decrease in So after 48 (A10 and H9C2(2-1) cells) or 72 (HepG2 cells) hr. Pretreatment (30 min) with SP600125 1) attenuated the FB1-induced Sa and So increases at 24 hr and maintained the Sa increase at 72 hr in HepG2 cells, 2) attenuated the FB1-induced Sa increase at 24 and 48 hr in A10 cells, and 3) had no effect on FB1 induced Sa and So changes in H9C2(2-1) cells. The results suggest that the interaction between the JNK signaling pathway and the sphingolipid metabolic pathway during FB1 exposure is transient and cell type specific. 
7 days, 
in the experimental setting. 
30 mg/kg/day. Nasal findings involved the respiratory and olfactory mucosa and were primarily located in the nasopharynx and ventral and lateral nasal cavity. Based upon the nature and distribution of the nasal changes, it was hypothesized that they were caused by local irritation associated with test material entering the nose from reflux and/or the dosing procedure. However, it was important to rule-out metabolism based nasal toxicity as had been observed in other studies (Pino et al, 1999). To test this hypothesis, rats were administered the test article by daily 8-hour intravenous (iv) infusions at doses up to 50 mg/kg/day for seven days (AUC(0–24 hr) values at 50 mg/kg/day were comparable to 30 mg/kg/day orally). No nasal tissue changes were observed in the iv study confirming the hypothesis that the findings in the oral study were related to reflux and/or the gavage dosing procedure. To further evaluate nasal effects related to oral gavage dosing, nasal cavities from control rats (n = 428) were reviewed from our database of oral toxicology studies. Findings attributable to reflux were observed in one study with a low-pH vehicle (phosphoric acid/maltitol), but not in studies using vehicles with a more neutral pH (e.g. deionized water or methylcellulose/polysorbate 80). In summary, when presented with rat nasal findings in oral toxicology studies, a differential etiology needs to include local irritation associated with oral gavage dosing. 
-Humanized Mice: Mechanism of the Differential Response to Peroxisome Proliferators
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