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P1 Altered Expression of Gene Products Involved as a Complex in the Endogenous Hepatocarcinogenesis of Rats Fed a Choline-Deficient, L-Amino Acid-Defined Diet
1 Sasaki Institute, Sasaki Foundation, Chiyoda, Tokyo, Japan Chronic feeding, for up to 2 years, of a choline-deficient, L-amino acid-defined (CDAA) diet results in a high incidence of hepatocellular carcinomas (HCCs) in male rats through endogenous mechanisms. Its morphological course consists of sequentially developed (pre)neoplastic and nonneoplastic liver lesions, and its underlying mechanisms are associated with oxidative stress, multiple signaling alterations, and genetic and epigenetic changes of specific genes. These characteristics are closely similar to those seen in human hepatocarcinogenic cases including those due to an infection of hepatitis viruses. Furthermore, this has recently been recognized as a good animal model for human nonalcoholic steatohepatitis (famous as NASH). The present study was conducted as a part of our work to explore details of molecular events occurring in this model with our aim to obtain information contributable to the control of human cancers, especially HCC, a deadly human neoplasm. Male Fischer 344 rats (6 weeks old) were fed the CDAA diet or a control diet for up to 70 weeks and serially sacrificed to obtain livers. Using the livers obtained up to the end of week 12, mRNA and protein expressions of 18 cytokines were assessed by a ribonuclease protection assay and an immunohistochemical technique, respectively. Using the livers obtained at the end of week 70, comprehensive gene expression profiles were assessed for HCCs, their surrounding tissues and the normal tissues by an oligonucleotide microarray technique for 3757 genes. All assessments were conducted using at least 5 samples per group from individual animals. mRNAs of the cytokines including IL-1-alfa, IL-1-beta, TNF-alfa, TGF-beta-1, TGF-beta-2, and TGF-beta-3 were overexpressed in differentially time-dependent manners in the early phase within the first 12 weeks. At the end of week 12, proteins of such cytokines were also overexpressed. IL-1-alfa and TNF-alfa proteins were overexpressed more remarkably in preneoplastic liver lesions than in their surroundings. IL-1-beta and TGF-beta-1 proteins behaved similarly, but the differences between inside and outside of preneoplastic lesions were less evident. TGF-beta-2 protein was overexpressed less remarkably in preneoplastic liver lesions than in their surroundings. TGF-beta-3 protein behaved similarly, but the difference between inside and outside of preneoplastic lesions was less evident. Comparing profiles among HCCs, their surroundings and normal liver tissues, 147 genes were differentially expressed, which were classified according to the expression patterns into 4 identical clusters by different methods. These contained conspicuous changes for notable genes interacting each other within a bio-signaling complex. Such genes included transcription factors (e.g., hepatic nuclear factor-1 [Tcf1], hepatic nuclear factor-4 and Rheb), signaling factors (e.g., IL-1 receptor, IL-2 receptor, PDGF receptor, beta-, NGF, FGF-5, FGF-9, and COX-1), apoptosis and cell proliferation regulatory factors (e.g., caspase 3, inhibin, lifeguard, c-fos, src-related tyrosine kinase, and Tsc2), pre-mRNA processing factors (e.g., cyclin L and zinc finger protein 265), metabolic enzymes (e.g., CYP1B1) and anti-oxidant defense system members (e.g., phospholipase C and superoxide dismutase). The present results indicate that altered expression of numerous gene products is involved as a complex in the endogenous hepatocarcinogenesis of rats fed the CDAA diet.
P2 Immunohistochemical Characterization of PCB- and Arsenic-Induced Hepatic PreneoplasiaColorado State University, Fort Collins, Colarodo, United States
Polychlorinated biphenyls (PCBs) and arsenic are common pollutants and are likely to be found in environmental mixtures. The coplanar PCB126 (3,3',4,4',5-pentachlorobiphenyl) exhibits dioxin-like promotional effects on hepatocarcinogenesis. Arsenic is a clastogen that has been suggested to act at the progression stage of carcinogenesis or as a cocarcinogen. This study evaluated mixtures of these two chemicals to determine potential interactions at the promotion and progression stages of carcinogenesis. Staining for glutathione-S-transferase (placental form) (GST-P) identifies promoted foci, while staining for transforming growth factor alpha (TGF This research is supported by National Institute of Environmental Health Sciences Grant #5 K08 ES00380.
P3 Drh1 Locus Play an Important Role in DRH Proneoplastic Stage of HepatocarcinogenesisKyoto University, Kyoto, Japan The DRH is an inbred rat strain established by selective mating of 3'-Me-DAB resistant progeny of closed colony Donryu rats over 20 generations (Higashi T et al.). Genetic analysis shows that 2 semidominant QTLs, Drh1, and Drh2, are responsible for strong resistance to chemical-induced hepatocarcinogenesis in DRH strain rats. To evaluate the effect of a single Drh1 locus on various stages of liver carcinogenesis, we constructed a congenic strain DRH-Drh1F/F by transferring a susceptible Drh1 allele of F344 to DRH rats by marker-assisted backcrossing. The congenic rats had a ~42 cM segment of chromosome 1 bearing Drh1, but the Drh2 was of the DRH allele. After oral administration of 3'-Me-DAB for 8 weeks, the DRH-Drh1F/F rats showed the intermediate data between F344 and DRH in fibrosis, enzyme-altered foci, GST-P expression, and proliferation of liver cells. In the liver of carcinogen-fed DRH rats, there was intensive apoptosis as detected by Tunel stain, but in the liver of F344 and congenic rats, it was significantly suppressed. Injection of lead nitrate (100 µmol/kg.b.w.) induced a wave of liver cell proliferation seen by BrdU uptake within a few days in F344 and congenic rats, but not in DRH rats. Drh1 seems to play an important role in an earlier stage of hepatocarcinogenesis, presumably by effective removal of initiated cells.
P4 Resolution of Activated Hepatic Stellate Cells by a Chinese Medicine Preparation, Han-Dan-Gan-Le
1 University of Louisville, Louisville, Kentucky, United States Han-Dan-Gan-Le (HDGL), a Chinese medicine preparation composed of Salvia miltorrhiza, Radix paeoniae, Astragalus membranaceus, Stephania tetrandra, and dried leaves of Ginkgo biloba, has been used successfully to treat human liver fibrosis and cirrhosis for years in China. However, the mechanism of action of HDGL is unknown. This study was undertaken to examine the effect of HDGL on the activity of hepatic stellate cells (HSC), a cell population responsible for collagen production and accumulation in the liver. Activated human HSC (passages 18–25) were cultured in DMEM containing 10% fetal bovine serum and antibiotics. Activation of the HSC was determined by immunostaining with antibody against á-smooth muscle actin and collagen I. Treatment with HDGL at a concentration similar to clinical application caused regression of the activated HSC along with increased lipid droplets and decreased production of collagen I. Analysis of gene expression revealed that HDGL caused down-regulation of collagen synthesis and TIMPs and up-regulation of collagenolytic enzymes in the HSC. Furthermore, cocultures of HSC and hepatocytes treated with HDGL displayed selective resolution of the activated HSC and stimulated proliferation of the hepatocytes. These results thus demonstrate that HDGL-induced fibrinolysis in humans may relate to the resolution of the activated HSC by the Chinese medicine preparation.
P5 Hydroxyurea(HU)-Induced Apoptosis in Fetal Mouse
1 The University of Tokyo, Tokyo, Japan Hydroxyurea (HU)-induced apoptosis in fetal mouse a ribonucleatide reductase inhibitor, induces morphological anomalies in the brain, craniofacial tissues, and limbs in experimental animals. In human beings, cases of neonatal respiratory distress, acute alveolitis, and skin lesion have been reported. In this study, we assessed the cytotoxicologic effects of HU on the fetal tissues by exposing pregnant mice to HU on day 13 of gestation. A moderate to marked increase in the number of pyknotic cells was observed in the brain and lung. A mild increase in the number of pyknotic cells was also found in the craniofacial mesenchymal tissues, limb buds, and so on. These pyknotic cells had nuclei positively stained by the TUNEL method and they also showed electron microscopic characteristics identical to those of apoptotic cells. In the telencephalus, the number of TUNEL positive cells began to increase at 3 hours after treatment, peaked at 12 hours, and rapidly decreased at 24 hours. On the other hand, TUNEL-positive reaction in the lung peaked at 6 hours. This positive reactivity for TUNEL in the lung was seen mainly in mesenchymal cells. The number of p53-positive cells peaked at 3 hours in both the brain and lung. As to the expression of p53 and its transcriptional genes mRNAs, there were some differences between the brain and lung. In the brain, a significant increase in the expression of fas, fasL, bax, mdm, and p21 mRNAs was detected. In the lung, the expression of p53, bax, apaf1, and p21 mRNAs was significantly elevated. These results suggest that p53 may play an important role in HU-induced apoptosis in the mouse fetal brain and lung.
P6 Specific Excision of the Selenocysteine tRNA[Ser]Sec (Trsp) Gene in Mouse Liver Induces Lethal Hepatocellular Apoptosis
1 SAIC Frederick, Frederick, Maryland, United States Selenium is essential in mammalian embryonic development. However, in adults, selenoprotein levels in several organs including liver can be substantially reduced by selenium deficiency without any apparent change in phenotype. To address the role of selenoproteins in liver function, mice homozygous for a floxed allele encoding the selenocysteine (Sec) tRNA[Ser]Sec gene were crossed with transgenic mice carrying the Cre recombinase under the control of the albumin promoter that expresses the recombinase specifically in liver. Recombination was nearly complete in mice 3 weeks of age, whereas liver selenoprotein synthesis was virtually absent, which correlated with the loss of Sec tRNA[Ser]Sec and activities of major selenoproteins. Total liver selenium was dramatically decreased, whereas levels of low molecular weight seleno compounds were little affected. Plasma selenoprotein P levels were reduced by about 75%, suggesting that selenoprotein P is primarily exported from the liver. Glutathione S-transferase levels were elevated in the selenoprotein-deficient liver, suggesting a compensatory activation of this detoxification program. Mice appeared normal until about 24 hours before death. Most animals died between 1 and 3 months of age. Death appeared to be due to diffuse hepatocellular apoptosis. Hepatocytes were positive with the TUNEL assay and DNA laddering was demonstrated in vitro. Mice had concomitant necrosis of peritoneal and retroperitoneal fat. These studies revealed an essential role of selenoproteins in liver function. Funded in part by NCI NIH contract NO1-CO-12400 and NIH grants GM061603 and GM065204.
P7 Hepatic Glycogen Nuclei in a Cynomolgus Monkey with Spontaneous Diabetes MellitusNerviano Medical Science, Milan, Italy
A 9-year-old female Cynomolgus monkey not currently on study presented with a history of weight loss over the previous 3 months. The animal’s appetite remained good and there were no other clinical signs or symptoms. The most significant clinical chemistry finding was a marked hyperglycemia (710 mg/ dL; laboratory range: 45–88 mg/dL) and hypertriglyceridemia (941 mg/dL; laboratory range: 28–79 mg/dL). Other, less dramatic, changes were seen in plasma concentrations of total cholesterol, liver enzymes, and urea. In addition, a significant lymphopenia was present (0.55 x 103/
P8 Porcine-Serum-Induced Hepatic Fibrosis in Brown Norway and Wistar RatsDepartment of Veterinary Pathology, The University of Tokyo, Tokyo, Japan Many hepatic fibrosis models have been developed, but detailed fibrogenic process and mechanisms of hepatic fibrosis are still obscure. Porcine serum (PS) induces hepatic fibrosis with little hepatocyte damage, while many models accompany prominent hepatic damage. The mechanisms of PS-induced hepatic fibrosis were examined in Brown Norway (BN) and Wistar rats. Intraperitoneally Thirty 6-week-old male rats each of BN and Wistar strains were used. The rats of each strain were divided into 2 groups. One group was injected ip with 0.5 ml sterile PS twice a week for up to 8 weeks and the other group was treated with physiological saline in the same way and served as controls. At 1, 2, 3, 4, and 8 weeks, 3 rats of each group were sacrificed by blood sampling from abdominal aorta under ether anesthesia at 24 hours after the last PS or saline injection. Liver, spleen, and serum were collected and relative organ weight (g%) was calculated. Liver and spleen, were fixed with 10% neutral buffered formalin and a part of each liver was frozen with liquid nitrogen. Four-micrometer paraffin sections were stained with hematoxylin and eosin. Total RNA was extracted from frozen liver and used for Affymetrix GeneChip microarray analysis and reverse transcription-polymerase chain reaction (RT-PCR). According to the results of microarray analysis and RT-PCR, immunohistochemical staining was carried out on frozen sections. Serum levels of PS-specific IgG1, IgG2a, IgA, IgM, and IgE were examined. Relative spleen weight increased in 2 strains but relative liver weight did not. Histopathologically, the development of PS-induced hepatic fibrosis differed between BN and Wistar rats, and formation of hepatic fibrosis occurred earlier in the former. In spleen, enlargement of white pulp was detected in PS-treated groups. In microarray analysis, the expression of genes linked to antigen presentation (CD74, major histocompatibility complex (MHC) class II alpha chain and beta chain) was elevated earlier in BN rats than in Wistar rats. The results of microarray analysis were confirmed by RT-PCR in BN rats, but not in Wistar rats. Immunohistochemically, the numbers of anti-PS, RT1.B-1, ED1, ED2, CD4, or CD8 positive cells increased earlier in BN rats than in Wistar rats. Serum IgG1 and IgG2a levels were elevated, while IgA, IgM, and IgE levels were not. CD 74 and MHC class II are well known to be closely related with exotic-antigen presentation and antibody production. Therefore, the present results suggest that antigen presentation is an important process in PS-induced hepatic fibrosis and that the difference in the development of hepatic fibrosis between BN and Wistar rats may be at least due to the difference in MHC class II-related antigen presentation. The increase in serum immunoglobulin levels may be induced by MHC class II-related antigen presentation, and spleen may be a main working organ in immunoglobulin production.
P9 Impaired Liver Regeneration in db/db Mice After 2/3 Partial HepatectomyThe University of Tokyo, Tokyo, Japan Ob/ob mice and db/db mice are congenitally deficient of leptin and leptin receptors, respectively. Both of them are known as animal models of obesity and diabetes mellitus and their livers show fatty change. In ob/ob mice, the liver regeneration is reported to be impaired after two-thirds partial hepatectomy, while it has not yet been examined in db/db mice. In this study, we performed two-thirds partial hepatectomy in db/db mice, and examined the liver weight recovery rate and bromodeoxyuridine labeling index of hepatocytes and non-parenchymal cells until 10 days after partial hepatectomy. Comparing with the results in lean +m/+m mice, the liver regeneration in db/db mice was impared. Wound healing is also reported to be impaired in db/db mice through the unbalanced mRNA-expression of vascular endothelial growth factor (VEGF), angiopoietin (Ang)-1 and -2. We also examined the possible role of these factors in the impaired liver regeneration by examining the protein expression of VEGF, Ang-1 and -2 in the liver of db/db and +m/+m mice after partial hepatectomy by Western blotting. The level of protein expression of Ang-1 and -2 was not changed significantly and clear peak was not observed during liver regeneration in both db/db and +m/+m mice. On the contrary, the protein expression pattern of VEGF was quite different between db/db and +m/+m mice. In +m/+m mice, the band of VEGF-164, the size of which is about 80 kD, appeared from 3 days after partial hepatectomy, while the expression of such band was not observed in db/db mice. Then, we examined the expression of VEGF mRNA in the liver of db/db and +m/+m mice after partial hepatectomy by RT-PCR. Interestingly, the expression level of VEGF mRNA was not significantly changed during liver regeneration in both db/db and +m/+m mice, suggesting that VEGF expression might be controlled by the posttranscriptional level. In conclusion, the decreased expression of VEGF protein is considered to be very important in the impaired liver regeneration in db/db mice, although it is not clear whether the decreased expression of VEGF protein in db/db mice was the primary event or a secondary event to the impaired hepatocyte proliferation. Harmonized proliferation of hepatocytes and nonparenchymal cells, including sinusoidal endothelial cells, necessary for the completion of liver regeneration is influenced by VEGF.
P10 Spontaneous Background Lesions in the Gall Bladder and Liver of the Common Marmoset (Callithrix Jacchus) on Toxicology StudiesInveresk, Edinburgh, Scotland, United Kingdom Histological lesions were evaluated in the liver and gall bladder of 64 apparently healthy common marmosets (Callithrix jacchus) from the control groups of toxicology studies of 1 to 13 weeks duration. The most common lesion in the gall bladder was multifocal or diffuse chronic active cholecystitis (35%). The most common lesions in the liver were diffuse glycogen vacuolation (100%), focal inflammation (61%), multifocal sinusoidal dilation (25%), and fibrous tags on the capsule surface (14%). There was a higher incidence of gall bladder lesions in females. Hepatic lesions were equally distributed between males and females. These results show a high incidence of background inflammatory lesions in the liver and gall bladder of young healthy marmosets, and that care should be taken when interpreting these findings in toxicology studies.
P11 Gene Expression in Regenerating Rat Liver Following Partial Hepatectomy: A Detailed Time CourseNational Institute of Environmental Health Sciences, Research Triangle Park, North Carolina, United States Following two-thirds partial hepatectomy (PH) in the rat, hepatocytes undergo compensatory hyperplasia to reestablish the original volume of the liver. Little was known regarding the initiation and progression of hepatocytes through this regenerative process until the advent of microarray technology. To elucidate further the mechanisms of liver regeneration, we used the in-house National Institute of Environmental Health Sciences rat cDNA microarray containing over 6,000 genes/ESTs to measure changes in hepatic gene expression in a detailed time-course study following PH. Age-matched, male Fisher-344 rats were euthanized at 2 hours and 4 hours and then at 4-hours intervals for 52 hours following PH; sham-operated controls were used at each time point for comparison. By 52 hours after PH, almost 70% of the liver volume was egenerated. A detailed review of the histopathology was completed along with immunohistochemistry using multiple proliferative markers (PCNA, MCM, and Ki67) showing 3 waves of proliferation. Microarray analysis detected genes whose expression patterns mirrored the waves of proliferation; many of the genes were already known to be involved in cell-cycle progression and cell growth. In addition, prior to the first proliferative wave at 24 hours, transcription and growth factors, which initiate and regulate hepatocytic growth and progression through the cell cycle, were overexpressed. These genes remained upregulated throughout the 52-hour time course. This application of histopathology, immunohistochemistry, and microarray technology has provided additional insights into the mechanisms underlying liver regeneration.
P12 Hepatotoxicological Differences with ET-743 in Sprague–Dawley Rats and Cynomolgus Monkeys
1 Johnson & Johnson Pharmaceutical Research and Development, Beerse, Belgium Yondelis (trabectedin, ET-743) is a tetrahydroisoquinoline compound isolated from the marine ascidian Ecteinascidia turbinata. The compound is currently under clinical investigation in phase II trials and has shown activity in ovarian, breast, and advanced pretreated soft tissue sarcoma. In patients, the most prevalent drug induced toxicities are noncumulative neutropenia, reversible increases in transaminase values and fatigue. In order to better understand the hepatotoxic potential of Yondelis its safety was evaluated in repeated dose studies in rodent (Sprague–Dawley rats) and nonrodent models (Cynomolgus monkeys). Cynomolgus monkeys were chosen as the preferred nonrodent species due to the similarities in metabolic profile to that of humans. In these studies, ET-743 was administered via a 3-hour intravenous infusion every 3 weeks for 3 (rat) or 4 (monkey) cycles, or weekly for 3 weeks followed by 1 week without treatment (3 cycles). After repeated administration in rats, mortality was observed at 50 µg/kg in females and at 50–75 µg/kg in males. A pronounced dose-dependent and only partially reversible hepatotoxicity (increased transaminases, hepatocytic necrosis, bile duct inflammation, and proliferation) was noted, and toxicity was cumulative and more pronounced in female rats. The difference in gender sensitivity in rats is likely to be linked to differences in the metabolic profile, biliary excretion and/or liver retention. Adminstration of a weekly 30 µg/kg-dose was better tolerated by Cynomolgus monkeys than dosing 70 µg/kg every 3 weeks. Hepatotoxicity was less pronounced than in rats and noncumulative in nature (increased transaminases, hepatocellular hypertrophy, hepatocytic degeneration, or necrosis and mixed inflammatory cells in the sinusoids and portal tracts). At 50 mg/kg, exposure to ET-743 was however higher in monkeys (23–28 µg · h/L) than in rats (1.7–8.6 µg · h/L), without gender difference. The similar profile of hepatic changes induced by chronic ET-743 treatment in humans and monkeys suggest that the Cynomolgus monkey is a more relevant and predictive model for human Yondelis hepatotoxicity than the rat.
P13 Magnetic Resonance Microscopy of Acetaminophen Hepatotoxicity in the Rat
1 North Carolina State University, Raleigh, North Carolina, United States Acetaminophen is a commonly used analgesic antipyretic drug that, in gross overdosage, causes severe centrilobular liver necrosis and is a frequent cause of acute liver failure in humans. Acetaminophen is a well-studied model hepatotoxicant that can also be utilized to assess the strength and weaknesses of genomics and proteomics technologies as toxicological tools. Previous studies have revealed that there is an unexplained and striking inter- and intralobular variability of acute hepatic necrosis with some regions having massive necrosis and adjacent areas within the lobe or other lobes from the same animal showing no injury at all. Magnetic resonance microscopy (MRM) is fast becoming a valuable tool to visualize istologic alterations in whole animals. MRM was applied in this study to determine (1) both the 2- and 3-dimensional patterns of the inter-and intralobe variability in hepatic necrosis and (2) if the sites of injury were located preferentially to specific vascular landmarks in rats treated with a high dose of acetaminophen. Six male Fisher 344 rats approximately 7–8 weeks of age (150–210 gm) were treated with a single dose of 2,000 mg/kg of acetaminophen by gavage, 3 control rats received 0.5% ethyl cellulose only by gavage. Under deep anesthesia (Nembutal and butorphanol, IP), animals were perfused via a transcardial approach first with heparinized saline to exsanguinate and then with a mixture of neutral buffered formalin and contrast agent (Magnevist). MRM imaging was performed at the Center for In Vivo Microscopy, a National Institutes of Health National Center for Research Resources (NIH/NCRR) and involved the use of a 7.0 Tesla magnet using a 3D spin warp encoding TR = 100 ms, TE = 5 ms. The MRM liver images for each were reviewed without knowledge of treatment. By MRM, all 6 treated rats had irregular pattern of mottled parenchymal alterations throughout most liver lobes that were not evident in the 3 control rats. Three-dimensional reconstruction of the hepatic venous vascular system revealed severely blunted proximal branches of the veins in treated rats indicating a treatment-related massive injury to the hepatic venules (central veins). Histologic H&E stained sections of each lobe confirmed severe hepatic centrilobular necrosis and vessel injury. Further studies are under way to make direct correlations of the MRM lesions with the histologic findings for each liver lobe (median, left, right, and caudate) and further assess the existence of intralobe variation. In conclusion, MRM is a valuable tool to detect a 3-dimensional view of the distribution and extent of hepatocellular necrosis and the relationship of the injury to the vasculature in the rat liver after treatment with the hepatotoxicant acetaminophen.
P14 Prediction for Human Hepatotoxins in a Cryopreserved Hepatocyte Test System via Transcriptional ProfilingMillennium Pharmaceuticals, Cambridge, Massachusetts, United States The prediction of development limiting toxicity in the late lead optimization using safety pharmacology studies and 5- to 7-day repeat dose toxicity studies in rat and higher species has significantly reduced attrition (associated with systemic toxicity) to 15% compared to recent benchmark standards (46% attrition from nonclinical toxicity and clinical side effects, Brugera, 2003). However, these activities consume substantial chemical synthesis resources, thereby engendering relatively high costs. The opportunity for the toxicology discipline to create a discontinuity in the efficiency of the drug discovery process exists in leveraging molecular technology in cell-based systems to predict for systemic toxicity. Biochemical endpoints including those for cell viability have not been demonstrated to have predictive value for systemic toxicity. The toxicology community has been slow to engage in in vitro test systems to predict for toxicity other than with genetic toxicity and hERG binding assays. Cell-based systems using microarrays have been demonstrated to predict successfully for pharmacology specific patterns of gene expression. A growing number of companies have focused on creating toxicity specific patterns in cell-based assays. Transcriptional profiling of a limited number of genes specific to toxicologic responses in cell-based systems represents a practical approach for screening late in HTL and early in LO. Simultaneously an improved limited gene set can be identified using a microarray from the same test system. Using transcriptional profiling of a few literature-based stress and cell cycle related genes in a cryopreserved human hepatocyte test system to predict for human hepatotoxicity enables some differentiation of hepatotoxins, especially the egregious hepatotoxins such as geldanomycin via up-regulation of several of the toxicity genes. Twenty drugs of known prototypical toxicity potential including drugs causing idiosyncratic injury have been evaluated in this testing paradigm including demonstration of repeatability. Additionally these same chemicals have been profiled with microarrays in search of additions to the current limited gene set.
P15 The Prediction of Hepatotoxicity Using DEREK for Windows
1 LHASA Limited, Leeds, United Kingdom Hepatotoxicity is a major cause of compound attrition within the pharmaceutical industry. Screening of compounds for hepatotoxicity allows potential problems to be identified and investigated early in the drug development process. Computer prediction of toxicity provides one means by which this screening can be achieved. DEREK for Windows (DfW) is a knowledge-based expert system for toxicity prediction. The knowledge base is composed of alerts, example compounds, and rules that may each contribute to the toxicity predictions made by the system. Alerts describe the relationship between a structural feature, or toxicophore, and the toxicological endpoint with which it is associated and are accompanied by a summary of supporting evidence, references, and a selection of example compounds and their relevant toxicological data. To date, no systematic attempt has been made to provide comprehensive coverage of hepatotoxicity within the DfW knowledge base. In an effort to improve the predictive performance of DfW for hepatotoxicity, therefore, relevant toxicity data from a range of sources have been collated. Currently, data have been collected for 1,139 compounds for which at least some evidence of hepatotoxicity has been reported either in humans and/or experimental animals. Chemical and toxicological data relating to these compounds have been stored in a structure-searchable database. Present efforts are focussed on the derivation of new and modified alerts for hepatotoxicity based on the compound classes in this database that are not currently correctly predicted by DfW. As part of this process, more detailed hepatotoxicity data are being gathered for compounds of interest using a standardised set of data fields developed for this purpose. These data fields are based on the recommendations of the hepatoxicity expert subcommittee of the ILSI/SAR database project that are also currently being used in the further development of LHASA Limited’s VITIC toxicity database.
P16 Endogenous Eosinophilic Crystal Formation Associated with Bronchial and Biliary Hyperplasia/Hypertrophy and Inflammation in Rotenone Treated Tg.AC and Wildtype FVB/N Mice
1 Department of Veterinary Pathobiology, College of Veterinary Medicine, University of Illinois, Urbana, Illinois, United States Eosinophilic crystals, similar to Charcot–Leyden crystals in humans, have been observed in the respiratory tract, biliary tree, and other tissues of C57BL/6, Swiss Webster, and related strains of mice as well as in genetically altered mice, particularly those generated on a C57BL/6 or Sv/129 background. Pulmonary crystals have been identified as Ym1 (T-lymphocyte-derived eosinophil chemotactic factor). They have been associated with acidophilic macrophage pneumonia. However little information is available on the morphology of the lesions associated with the crystals. Seventeen 24- to 37-week-old, female: Tg.AC and FVB/N mice with myeloproliferative disorder and acidophilic macrophage pneumonia that developed following rotenone treatment were examined (see Heinz-Taheny et al., this meeting). Extracellular eosinophilic crystals were observed in both lung and liver of 10/17 mice while 4/17 had crystals only in lung and 1/17 only in liver. The eosinophilic crystals were needle shaped, deeply eosinophilic, and refractile. In the lung, the crystals were found primarily in the lumen of the large hilar bronchi, although they were occasionally seen in bronchioles and in the alveolar duct. Typically, the hilar bronchial epithelium was hyperplastic with hypertrophic epithelial cells containing intracytoplasmic deeply eosinophilic globules and occasionally crystals. The submucosa, and occasionally the epithelium, was infiltrated by mixed inflammatory cells. Lungs from all mice contained variable numbers of macrophages, primarily in the alveoli, but also occasionally in the airways (acidophilic macrophage pneumonia). The macrophages had abundant cytoplasm that contained fine eosinophilic crystals. Multinucleated giant cells were often present in alveoli. In the liver, the eosinophilic crystals were present within scattered bile ducts that were lined by hyperplastic and hypertrophic epithelium that was filled with large eosinophilic globules. Many of the affected bile ducts appeared to have submucosal glands and were surrounded by connective tissue and mixed inflammatory cells. In summary, eosinophilic crystals in both bronchi and bile ducts were associated with the presence of eosinophilic intracytoplasmic globules within hyperplastic/hypertrophic epithelium as well as inflammation in surrounding tissue. Both Tg.AC and the wild-type FVB/N mice developed similar lesions.
P17 Eosinophilic Crystals in the Inflamed Lungs of Three RatsApplied Veterinary Pathobiology, PLLC, Bainbridge Island, Washington, United States Eosinophilic crystals forming endogenously from the YM protein family have been reported in respiratory (YM-1) and gastrointestinal tracts (YM-2) and other tissues of mice on toxicology and experimental studies, but these crystals have not been reported in rats. This case report describes the histological identification of a small number of eosinophilic crystals in the lungs of 3 Sprague–Dawley rats in the control groups of 2 safety studies of 13 weeks’ and 6 months’ duration. In both studies, vascular infusion via chronic implants resulted in mortality and morbidity from bacterial contamination. Bacterial colonies were readily identified in the lungs of rats that died or were euthanatized, but bacteria were infrequent in lungs collected at study termination. Of 40 rats in the saline vehicle control groups (10/sex/study), 6/8 survivors at 13 weeks, and 7/13 rats after 6 months had vascular injury restricted to the lungs. The lung lobes contained varying numbers of blood vessels with chronic-active perivasculitis and vasculitis, and thrombosis with sporadic recannulation in the 6-month study. Alveolar walls adjacent to these vascular changes were frequently hypercellular, and alveolar/foamy macrophages were present within alveoli. Additionally, the lungs from 1/6 affected rats at termination at 13 weeks, and 2/7 affected rats at termination at 6 months had 1–2 foci of extracellular and rarely intracellular eosinophilic crystals associated with activated alveolar macrophages. Although the number of crystals was small in the 3 rats (1 male, 2 females), the morphology was similar to eosinophilic crystals in the lungs of mice. These crystals in the rat lungs were not related to precipitation of a therapeutic and were distinct from cholesterol clefts and eosinophilic material found in alveolar histocytosis and lipoproteinosis. Detailed characterization of inducible proteins in the lungs of rats will be required to determine if these eosinophilic crystals are equivalent to YM-1 derived crystals in mice. However, a previously reported silicotic induced pulmonary macrophage factor or bronchoalveolar lavage protein-p58 (iSPMF p58 or iSBLP58) that has sequence homology to other members of the mammalian chitinase family including YM-1 (mouse lung), cartilage gp39 (human), and mammalian oviduct-specific protein suggests that a YM-1-like protein exists in the lungs of rats. Similar to YM-1 crystals in the lungs of mice, the crystals in the 3 rats in these studies were found in association with alveolar macrophages and secondary to lung injury. Although eosinophilic crystal formation is rare, rats appear to have a lung protein capable of autocrystalization during inflammatory events that is similar to the YM-1 protein in the lungs of mice.
P18 Two Cases of Localized Pulmonary Histiocytic Sarcomas in Beagle DogsMedicinal Safety Research Laboratories, Sankyo Co., Ltd., Fukuroi, Shizuoka, Japan Malignant histiocytic neoplasms derived from macrophages or dendritic cells have been recently diagnosed as cases of histiocytic sarcoma (HS) in dogs. Canine HS is classified as localized or disseminated HS according to the proliferative patterns. Localized HS arise from a single site, whereas disseminated HS is a multisystem disease previously described as malignant histiocytosis. Two cases of localized HS were found in the lung of untreated beagle dogs bred in our facility. Macroscopically, a distinct whitish nodule (approximately 10 mm in diameter) was observed in 1 lobe of the lung, but no such lesions were observed in the other organs or tissues in an 11-year-old male dog (case 1). Similarly, a distinct nodule (70 x 30 x 20 mm) was observed in the left caudal lobe of the lung but no such lesions were observed in the other organs or tissues in a 15-year-old male dog (case 2). Microscopically, both nodules were composed of neoplastic cells proliferating or infiltrating in a sheet-like fashion within The alveolar lumen with little destruction of the alveolar septa. These cells showed moderate-to-severe pleomorphism and atypia and had abundant eosinophilic to foamy cytoplasms. Erythrophagocytosis and multinucleated cell formation were observed in some of the cells. The neoplastic cells had the morphological characteristics of histiocytic cells. Large necrotic areas and invasion of blood vessels were also observed in case 2, which suggested that the lesion in case 2 was more malignant than that in case 1. In the immunohistochemistry and lectin histochemistry examinations, the neoplastic cells were positive for lysozyme, alpha1-antitrypsin, concanavalin A (ConA), ricinus communis agglutinin-1 (RCA-1), wheat germ agglutinin (WGA), and soybean agglutinin (SBA), but were negative for epithelial markers such as cytokeratin AE1/AE3 and cytokeratin MNF116. Proliferation cell nuclear antigen (PCNA) and Ki-67 (MIB-1) immunostaining demonstrated high cell proliferation activities in the neoplastic cells. Ultrastructurally, the neoplastic cells contained lipid droplets, primary lysosomes, and phagosomes. Based on the above findings and according to The WHO International Histological Classification of Tumors of Domestic Animals, 2nd series (2002) the present 2 cases were diagnosed as localized HS of the lung. This tumor is considered to rarely occur in dogs.
P19 Short-Term Cardiotoxic Damage Induced by Bis(2-chloroethoxy)Methane in the F344 Rat Revealed Light and Electron MicroscopicallyNational Institute of Environmental Health Sciences, Research Triangle Park, North Carolina, United States Bis(2-chloroethoxy)methane (CEM), a synthetic organic compound, is the starting agent from which polysulfides used in sealant applications are derived. Because of its high production, widespread usage, and metabolic conversion to thiodiglycolic acid, known to cause mitochondrial dysfunction and cardiotoxicity in rodents, it may pose a risk for the development of heart damage in humans. In this investigation, cardiotoxicity induced by 2-, 3-, 5-, and 12-day dermal administration of 400 and 600 mg/kg/day of CEM to F344 male and female rats was characterized. For all time points, hematoxylin and eosin-stained sections were examined to grade lesions; Masson’s trichrome stain was applied to reveal necrosis and fibrosis; immunohistochemical staining for troponin T was employed to indicate loss of myofiber integrity; and cell death was visualized by the TUNEL assay. The severity and incidence of lesions were similar among males and females and all 3 regions of the heart examined (atrium, ventricle, interventricular septum); damage consisted of time-related development of myofiber vacuolation, necrosis, mononuclear-cell infiltration, fibrosis, and atrial thrombosis. Changes were pronounced at day 2, increased in severity at day 3, appeared to decrease at day 5, and resolved by day 16, which corresponded to the 12-day dosing. After the initial damage induced by BCEM, or its metabolite, thiodiglycolic acid, the heart apparently launched protective mechanisms enabling it to cope with continued exposure to this toxicant while eliminating some damaged myofibers and reducing the damage by day 16. Ultrastructural analysis of 2- and 5-day-high-dose-treated females revealed the primary subcellular target, the mitochondrion, and 2 types of vacuolation, one that formed as damaged mitochondrial cristae disintegrated and bounding double membranes became reduced to singleness, and the other manifested as distention of the sarcoplasmic reticulum.
P20 Acute Hemorrhagic Micro-Infarction in Hearts of Rats Exposed to Ephedrine and Caffeine in CombinationNational Institute of Environmental Health Sciences, Research Triangle Park, North Carolina, United States Ephedra, also called ma huang, is a naturally occurring botanical derivative used in dietary supplements, whose active ingredients include ephedrine and guarana-derived caffeine. Because of the possible side effects of herbal medicines containing ephedrine and caffeine, including increased risk of stroke, myocardial infarction, and sudden death, the Food and Drug Administration recently banned the sale of ephedrine-containing products, specifically over-the-counter dietary supplements. Our objective was to investigate the cardiac pathology of ephedrine (25 mg/kg) and caffeine (30 mg/kg) administered to 7-and 14-week-old male F344 rats by oral gavage for 1 or 2 days. The ephedrine and caffeine exposure was approximately a 12- and 1.4-fold increase above the average human exposure based on a mg/m2 body surface area comparison. To confirm the nature of morphological changes and clarify their pathogenesis, we used a battery of histochemical (Barbebeito-Lopez Trichrome) and immunohistochemical (caspase-3, and anti-phospo-H2A.X) stains for myofiber coagulative necrosis and apoptosis, respectively. The 14-week-old rats were more sensitive to treatment, as some died within a few hours of dosing, in contrast to the 7-week group in which no deaths occurred. Several of the ephedrine/caffeine-treated rats died or were sacrificed in extremis 4–5 hours after the first dosing. The rest of the animals were sacrificed 4 hours after the second dosing. In the hearts of animals found dead or sacrificed 4–5 hours after the first dosing, changes were observed chiefly in the interventricular septum and, to a lesser extent, in the left and right ventricular walls. Massive interstitial hemorrhage, associated with degeneration of the surrounding myofibers, occurred at the subendocardial myocardium of the left ventricle and interventricular septum. Immunohistochemical detection of cleaved-caspase-3 revealed a multifocal generalized positive staining of the myofibers frequently localized in the interventricular septa. Other changes included multifocal, generalized myofibrillar loss associated with macrophage infiltration and hematoxylin-stained hyperbasophilic fragments of nuclei that were markedly positive for anti-phospho-H2A.X, a histone variant that becomes phosphorylated during apoptosis. The Barbebeito-Lopez Trichrome histochemical method, applied to indicate early myocardial necrosis, revealed generalized patchy yellow myofibers, showing cytoplasmic homogenization and loss of striation. This method proved most effective in clearly identifying morphologically altered myofibers indistinguishable by routine hematoxylin-and-eosin staining. In animals of the same dose group but terminated after the second dosing, the foci of myocardial degeneration and necrosis were already infiltrated by mixed inflammatory cells. This observed heart damage suggests that microinfarction constituted the lesions and consequently implies that necrosis occurred secondarily to ischemia. We hypothesize that a combined oral exposure to ephedrine and caffeine induces intense diffuse vasoconstriction of the coronary arterial system, decreasing myocardial perfusion and leading to degeneration and death of the cardiomyocytes. The findings from these rat-model studies show that ephedrine and caffeine administered together can cause cardiotoxicity.
P21 5-Hydroxytryptamine (5HT) Receptors in the Heart Valves of Cynomolgus Monkeys and Sprague–Dawley RatsGlaxo-SmithKline Inc., Research Triangle Park, North Carolina, United States Valvular heart disease (VHD) associated with the activation of serotonin [5-hydroxytryptamine (5HT)] receptors and/or increased circulating 5HT levels has been described in humans with carcinoid tumors and use of 5HT2b agonists, such as fenfluramine (Pondimin), dexfenfluramine (Redux), and ergot alkaloids (ergotamine and methylsergide). A similar association between pergolide (Permax) and VHD has been reported, also proposed to be mediated via the 5HT2b receptor. 5HT2b-receptor stimulation is known to cause fibroblast mitogenesis, and 5HT2b mitogenic signaling has been proposed to trigger the subendothelial cell proliferation and increased extracellular matrix in VHD-patients. In this study, we describe the mRNA expression levels and immunohistochemical localization of 5HT2b, 5HT2c, and 5HT1b receptors in the normal heart valves of male Cynomolgus (Cyno) monkeys and Sprague–Dawley (SD) rats. Four heart valves, namely mitral, tricuspid, semilunar, and pulmonary valves were collected from 2 monkeys (5 or 8 years of age) and 8 adult rats. We used real-time polymerase chain reaction (TaqMan) to quantify transcript levels of 5HT2b, 5HT2c, and 5HT1b receptors and immunohistochemistry to detect the tissue localization of these receptors. Each individual heart valve (valve leaflet) was snap-frozen in liquid nitrogen for TaqMan, and for immunohistochemistry, heart valves were fixed in 10% neutral buffered formalin (NBF). For rats, because of the small sample size, individual leaflets of each heart valve were pooled from 4 rats for TaqMan, and hearts from another 3 rats were fixed intact in 10% NBF for immunohistochemistry. In both species, positive immunostaining was noted for all 3 5HT receptors in mitral, tricuspid, semilunar, and pulmonary valves. However, the cell types showing positive staining were variable among the 3 5HT receptors: 5HT2b and 5HT1b were expressed in both endothelial cells as well as valvular interstitial cells, while 5HT2c was expressed only in the endothelial cells lining the valvular leaflet. In both species, immunohistochemistry results were correlated with 5HT2b and 5HT1b receptor transcripts (mean copies per 50 ng of total RNA) for all 4 valves. However, 5HT2c transcripts were lower than 5HT2b or 5HT1b in all cyno-valves, whereas 5HT2c transcripts were below the level of detection in any of the rat valves. Our data showed the expression of 5HT2b, 5HT1b, and 5HT2c in the normal heart valves of Cyno monkeys and SD rats. 5HT receptor immunohistochemistry and TaqMan techniques may be used to screen the potential valvular effects of compounds with 5HT2b receptor agonist activity.
P22 Mitochondrial Damage Revealed by Morphometric and Semiquantitative Analysis of Mouse Pup Cardiomyocytes Following In Utero and Postnatal Exposure to Zidovudine and LamivudineNational Institute of Environmental Health Sciences, Research Triangle Park, North Carolina, United States Zidovudine (ZDV), an antiretroviral drug used alone or in combination with other antiretroviral agents to treat HIV-infected pregnant women and their newborn infants, effectively reduces mother-to-child transmission of the virus. That myopathy and cardiomyopathy, related to mitochondrial damage, develop in some adults chronically treated with ZDV has long been known. Recent reports have suggested that similar adverse effects may occur in some infants exposed perinatally to ZDV. Using a mouse model of human neonatal exposure, we treated CD-1 pregnant mice twice daily with doses of 75 mg/kg ZDV plus 37.5 mg/kg lamivudine (3TC) throughout gestation and lactation. Pups were exposed by direct gavage beginning on postnatal day (PND) 4 and sacrificed on PND 28. Hearts were removed swiftly, and ventricles were processed for electron microscopy. This combination of ZDV and 3TC induced alterations in cardiomyocytic mitochondria in these mouse pups exposed in utero and neonatally. We observed clusters of damaged mitochondria more frequently in treated animals than in controls; damage included fragmentation and loss of cristae resulting in electron lucent matrices, occasional simple or whorled lamellated membranes within a mitochondrion, occasional rupture of the bounding double membrane, and rare apparent fusion of adjacent mitochondria. Morphometric and semiquantitative morphological analyses were performed on 3 micrographs from each of 3 blocks from each of 3 females and 3 males from the control and treated groups. Following independent visual assessment of mitochondrial damage in the 54 electron micrographs by 4 observers, all micrographs were scanned electronically and evaluated morphometrically for number and size of mitochondria. Treated mice showed significant increases in the mean area and decreases in the mean number of cardiomyocytic mitochondria compared to controls. These results may model cardiac damage reported in human infants similarly exposed to ZDV. Critical insights derived from animal-model data such as these may be used to mitigate risks to thousands of human infants receiving essential lifesaving therapy with antiretroviral drug
P23 A Photographic Spectrum of Nonneoplastic Renal Tubule and Transitional Epithelial Structures within Selected Renal Neoplasms of the Rat
1 Experimental Pathology Labs., Inc., Research Triangle Park, North Carolina, United States The identification and characterization of altered renal tubule and transitional epithelial structures are important in the diagnosis of several tumors of the rat kidney: Renal Mesenchymal Tumor (RMT) and Nephroblastoma (NB). Controversy still exists over the interpretation of these structures. Some pathologists consider these structures to represent nonneoplastic, preexisting and entrapped renal tubules or transitional epithelium that have undergone hyperplastic and/or metaplastic (embryonal) transformation. Other pathologists consider these structures to represent a neoplastic component of the tumor. Indeed, there are some tumors in which these structures represent a challenging and often puzzling dilemma for the pathologist. We present a number of microphotographs that illustrate the spectrum of changes that we encountered during a review of a large database (National Institute of Environmental Health Sciences/NTP) of RMT and NB. Our diagnostic criteria and previous observations of the experimental sequential development of RMT and NB contend that these structures represent a hyperplastic to metaplastic transformation. Although the mechanism for these changes are not known, the unique relationship during renal development between the primitive metanephric mesenchyme and the epithelial structures derived from the ureteric bud (derived from the Wolffian duct) might support a hypothesis that these structures are recapitulating embryologic histogenesis.
P24 Immunostaining Characteristics of Neuroendocrine and Cribriform Kidney Tumors in the TRAMP Mouse
1 Integrated Laboratory Systems, Inc., Research Triangle Park, North Carolina, United States The TRAMP mouse model of prostate cancer is a transgenic mouse strain created by insertion of the rat probasin (PB) gene fused to the SV40 T antigen (Tag) into C57Bl/6 mice. We previously reported that neuroendocrine neoplasms arise in the ventral prostate gland and urethra in the TRAMP mouse and also that cribriform pattern tumors arise in the kidney of both male and female TRAMP mice. Both the prostate/urethral tumors and the kidney tumors show positive immunostaining for SV40, suggesting they may be probasin-driven. In order to document the neuroendocine staining characteristics of the prostatic and urethral tumors and to investigate whether the cribriform pattern kidney tumors are of neuroendocrine origin, we have stained representative examples of these neoplasms with antibodies to the neuroendocrine markers, synaptophysin, chromogranin A, neuron specific enolase (NSE), and serotonin, and also with antibodies to cyclo-oxygenase-2 (COX-2), and androgen receptor. Slides were previously prepared when blocks containing the prostate were step-sectioning to obtain slides for grading of prostate lesions, the immunostaining was therefore performed on slides that had been archived for approximately 2 years. Under these conditions, synaptophysin was the most effective neuroendocrine marker, with antibodies showing highly sensitive and specific staining of both prostatic and urethral neuroendocrine tumors, but no staining of prostatic epithelium. Likewise, antibodies to NSE-stained neuroendocrine tumors specifically. Antibodies to chromogranin A and serotonin did not differentiate neuroendocrine tumors from prostatic epithelium. Antibodies to COX-2 did not stain either neuroendocrine tumors or prostatic epithelium. Antibodies to androgen receptor stained prostatic epithelium and to a lesser extent, neuroendocrine tumors. Cribriform pattern kidney tumors did not stain for synaptophysin or NSE. We conclude that synaptophysin is a specific and sensitive marker of neuroendocrine tumors in the TRAMP mouse and is effective on archived slides; also that the cribriform tumors arising in the kidney of these mice are not of neuroendocrine origin. This work was supported by National Institute of Environmental Health Sciences [N01- ES-35513].
P25 Renal Toxicity in Rats Treated with Doxorubicin Correlates with Clusterin ExperssionSchering-Plough Research Institute, Lafayette, New Jersey, United States Identification of sensitive and early biomarkers of renal toxicity are important as alterations in blood urea nitrogen (BUN) and serum creatinine only occur after substantial renal injury. Recent gene profile studies in nonhuman primates have indicated that clusterin is a potential biomarker for renal toxicity. This study investigated whether kidney clusterin expression is altered by anthracyclines in rodents. Anthracyclines are anti-cancer chemotherapeutics whose clinical use is often limited by their nephrotoxicity. Male Sprague–Dawley rats were treated with weekly intravenous doses of doxorubicin hydrochloride (either 1.0 or 4.0 mg/kg). Groups of rats were sacrificed at various times (5, 14, or 28 days) after administration of doxorubicin and compared to control groups for hematology, clinical chemistry, urinalysis, histopathological evaluation of the kidney, and immunohistochemical expression of clusterin. Clinical pathology parameters consistent with hyperlipidemic nephropathy were observed in a dose-dependent manner and included nonregenerative anemia, hypoalbuminemia, hypercholesterolemia, elevated triglycerides, with only minimally elevated BUN and creatinine. Histopathological lesions in the kidneys included varying degrees of nephrosis and tubular casts in a dose-dependent manner. Immunohistochemical staining for clusterin with a rabbit anti-human polyclonal antibody demonstrated increased expression in the tubular epithelium with dose-dependent intensity. These results demonstrate that clusterin protein expression correlates with drug-associated nephrotoxicity and that clusterin is a potential biomarker for renal damage.
P26 Exocrine Pancreatic Pathology in Female Sprague-Dawley Rats Following Chronic Treatmentwith 2,3,7,8-Tetrachlorodibenzo-p-Dioxin and Dioxin-Like Compounds
1 National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina, United States The National Toxicology Program (NTP) has recently conducted multiple 2-year bioassays in female Harlan Sprague–Dawley (HSD) rats to evaluate the chronic toxicity and carcinogenicity of dioxin-like compounds (DLCs), structurally related PCBs, and mixtures of these compounds. Here we describe the incidences and morphological aspects of pancreatitis and exocrine pancreatic cancer observed in the 2-year toxicity and carcinogenicity studies. Animals were treated by gavage for up to 2 years with 2,3,7,8-tetrachlorodibenzo-pdioxin (TCDD), 3,3',4,4',5-pentachlorobiphenyl (PCB), 2,3,4,7,8-pentachlorodibenzofuran (PeCDF), or a mixture (1:10:2 respectively) of these agents ("TEF Mixture"); control animals received corn oil-acetone (99:1) vehicle alone. A complete necropsy was performed on all animals, and a full complement of tissues was collected and examined microscopically. Increased incidences of several nonneoplastic changes in the exocrine pancreas, including cytoplasmic vacuolation, chronic active inflammation, atrophy, and arteritis were associated with treatment in each of the 4 studies. An increasing trend in incidence of pancreatic acinar cell adenoma/carcinoma was observed in the TCDD study. Low incidences of pancreatic acinar adenoma and carcinoma were also seen, in the PC126, PeCDF, and TEF-mixture groups. In the DLC-treated HSD rat, these neoplasms consisted of tubular and acinar structures. By comparison, 80% of all pancreatic neoplasms in humans are ductal in origin (ductal adenocarcinomas). There was only a single control animal (out of 207) with a pancreatic acinar cell neoplasm (an adenoma), confirming that this tumor is relatively uncommon. While the low incidences in these studies would not be particularly noteworthy when considered individually, the consistency of the effects in all 4 studies in female HSD rats for a relatively uncommon neoplasm suggest that these increases may be related to treatment. These studies indicate that the pancreatic acini are target tissues for dioxin and certain dioxin-like compounds.
P27 Gene Expression Can Differentiate Carcinogenic from Noncarcinogenic Doses of Dimethylarsinic Acid (DMA(V)) in the Transitional Epithelium of the Urinary Bladder from Female F344 RatsNational Research Council/U.S. EPA, Research Triangle Park, North Carolina, United States Arsenic is an environmental concern worldwide, and drinking arsenic contaminated water has been associated with increased incidences of skin, lung, and bladder cancer. Dimethylarsinic acid (DMA(V)) is a major metabolite of inorganic arsenic in rodents and humans and is the predominant form detected in the urine. Cytotoxicity and increased cell proliferation are associated with a dose dependent increase in tumors that arise from the cells that line the urinary bladder following chronic exposure to DMA(V) in the diet or drinking water in F344 rats. Since these arsenic-induced neoplasms arise from the transitional epithelium of the urinary bladder, gene expression profiling of this target cell population was performed to understand the molecular events associated with arsenic-induced carcinogenicity following exposure to DMA(V). Female 344 rats were exposed to 1, 4, 40, or 100 ppm DMA(V) in their drinking water for 4 weeks, RNA extracted from the transitional epithelium and labeled with Cy3 and Cy5 and hybridized to a rat oligonucleotide microarray (4,300 genes). Hybridizations were performed 4 times using independent total RNA preparations from four different animals in each dose group to ensure reproducibility. Differentially expressed genes were identified based on a 2-fold cut-off up or down and statistical (present in all samples, 1-way ANOVA, p < 0.01) significance. A companion set of rats were similarly exposed and the bladders were processed for routine light and transmission electron microscopic analysis. Ultrastructural changes were present after treatment with 40 and 100 ppm and were characterized by cellular hypertrophy, increased numbers of cytoplasmic vacuoles, and decreased numbers of mitochondria. Analysis of the gene expression data resulted in a total of 481 altered genes, among them 336 transcripts were induced and 145 were suppressed. Altered expression of genes that control apoptosis (Bad, Bax Pdcd2), cell proliferation (Btg1, Ncl, Vegf), cell cycle regulation (Tgfá, Igf2, Gadd45a), metabolism (Cyp2c, Cyp2e1, Cyba) and oncogenesis (Rap7a, Rabac1, Rab7) were observed after exposure to DMA(V) at any dose. Changes in oxidative stress response genes (Txn, Txn2, Glrx1, Gpx1, Sod1) were induced, consistent with the reported role of reactive oxygen species in DMA(V) induced toxicity of the rat bladder. Based on a greater than 3-fold expression in low dose (1 and 4 ppm) and high dose (40 and 100 ppm) groups compared to control animals, 2 sets of genes were identified that allow the differentiation between carcinogenic and noncarcinogenic doses of DMA(V). These data suggest that together with changes in expression of genes in the cancer control pathways, cellular toxicity that is present at the higher carcinogenic doses is also necessary to drive the DMA(V)-induced cancer process. (This is an abstract of a proposed presentation and does not necessarily reflect EPA policy).
P28 Retrospective Study of Background Testicular Pathology in Young Adult Hamsters Used in Preclinical Toxicity Studies
1 Huntingdon Life Sciences, East Millstone, New Jersey, United States The hamster is not routinely used in preclinical toxicology studies but its use may be necessary where pharmacokinetic considerations preclude the use of the rat and mouse. Due to its limited use, most laboratories have limited historical background pathology data for this species and there are few published reports of naturally occurring spontaneous pathology of the young hamster in the literature. Histopathological evaluation of the male reproductive tissues is further complicated by the fact that the hamster is a seasonal breeder and subject to cyclical degenerative and regenerative changes in spermatogenesis resulting from inherent circadian rhythms or changing photoperiodic regimes. To evaluate the range of background pathology in the testes and epididymides of young male Syrian hamsters, control animals from preclinical studies of 7 days to 13 weeks duration were used. Sections of hematoxylin and eosin stained testes and epididymides from 113 hamsters ranging in age from 8–21-week-old were examined microscopically and the incidence and severity of histopathologic abnormalities were recorded. Organ weight data was also evaluated. The most frequently observed microscopic findings in the testes were: tubules with complete germ cell loss (atrophic tubules) (22%), tubules with partial germ cell loss (hypospermatogenesis) (60%), and tubular dilatation (88%). Exfoliation of testicular germ cells and cell debris in the head and tail of the epididymis was present in most animals (96%). In general the testicular changes affected <25% of tubules (graded minimal to slight) but occasionally the changes were more extensive. In one study all testes were markedly reduced in weight and showed all the characteristics of seasonal dormancy. This occurred despite the fact that the animals were maintained on a long (12-hour light: 12-hour dark) photoperiod throughout the 13-weeks study. This data provides a profile of the types and incidences of incidental testicular changes occurring in the hamster. This information can be important when evaluating changes in test article treated animals.
P29 Epithelial-Stromal Tumor of the TRAMP Seminal Vesicle
1 National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina, United States The TRAMP (TRansgenic Adenocarcinoma Mouse Prostate) model, designed for researching human prostatic cancer, was genetically engineered to harbor a transgene composed of the simian virus 40 large-T antigen (SV40 Tag) promoted by the rat probasin gene. In addition to prostatic neoplasms, the TRAMP mouse develops tumors in the seminal vesicles. Rarely seen in the B6C3F1 mouse used in NTP studies, primary seminal vesicular neoplasms have been classified as adenoma, carcinoma, histiocytic sarcoma, sarcoma NOS, and undifferentiated carcinoma. However, this type of tumor in the TRAMP mouse cannot be placed in any of these categorizations. The present study was conducted to evaluate the pathology and histogenesis of TRAMP seminal vesicle neoplasms. TRAMP mice were necropsied at 11, 20, 24, 28, 38, or 40 weeks of age, and accessory sex organs were fixed in 10% neutral buffered formalin. Some animals were treated with 5-bromo-21-deoxyuridine (BrdU) for 3 days prior to euthanasia. Tissues were stained with hematoxylin and eosin, Masson’s trichrome, desmin, BrdU, and SV40 Tag. Tumors appeared in the neck and body of the seminal vesicles, but not in the tail, and large lesions sometimes obstructed secretion and resulted in swelling of the organ. Neoplastic stromal cells emerged multicentrically just beneath the epithelium of the preexisting anastomosing glandular architecture, with the stromal cells often densely packed between the epithelium and the smooth muscle layer. The stromal cells were spindle-shaped with small oval nuclei. They frequently exhibited mitotic figures and BrdU incorporation, SV40 Tag protein expression in the nuclei, and immunopositivity for desmin. The proliferative mesenchymal cells were lined by cuboidal to columnar glandular epithelium. Although the epithelium covering the stromal cells was negative for SV40 Tag and showed only occasional incorporation of BrdU, the epithelial component clearly participated in this biphasic proliferation, forming papillary, cystic, and tubuloglandular structures. Some of the larger papillary, polypoid lesions exhibited a phyllodes pattern resembling that seen in mixed epithelial-stromal tumors of the breast, prostate, and seminal vesicles of humans. These seminal vesicular tumors developed as early as 14 to 20 weeks, later than the prostatic tumors, which occurred as early as 10 weeks. Phyllodes lesions were observed at 28 weeks of age or later. No conclusive evidence of malignancy, such as invasion or metastasis, was identified. In summary, the seminal vesicular tumors were composed of neoplastic mesenchymal cells induced by the SV40 Tag oncoprotein and accompanied by proliferation of an epithelial-cell lining. Our recommended diagnosis is epithelial-stromal tumor. To our knowledge, similar tumors have not arisen spontaneously in conventional rodent strains.
P30 Epididymal Sperm Granuloma Induced by Chronic Administration of 2-Methylimidazole in B6C3F1 MiceNational Institute of Environmental Health Sciences, Research Triangle Park, North Carolina, United States The imidazole derivative 2-methylimidazole (2-MI), widely used in the manufacture of a variety of industrial products, has been identified as a by-product in several foods and detected in tobacco smoke. Because of the high potential for human exposure and a lack of carcinogenicity studies in rodents, a 2-year bioassay study was conducted by the National Toxicology Program (NTP). Those results from male and female F344/N rats and B6C3F1 mice revealed that epididymal sperm granulomas (SGs) occurred only in the male mice in a dose-dependent fashion. The present retrospective study was conducted to reevaluate the pathology and histogenesis of the SGs in the epididymis of mice treated with 2-MI. Groups of 60 male F344/N rats were fed diets containing 0, 300, 1,000, or 3,000 ppm 2-MI for 106 weeks, and groups of 60 male B6C3F1 mice were fed diets containing 0, 625, 1,250, or 2,500 ppm 2-MI for 105 weeks. Ten male rats and 10 male mice of each group were necropsied at 6 months. Testes (both sides) and epididymides (usually one side) of rats and mice were histopathologically reexamined. In mice, no differences occurred in mortalities between the control and treated groups. 2-MI induced SGs of the epididymis in mice at 2 years in a dose-related manner (0/50, 0/50, 3/50, 7/50, respectively). Histologically, most of the SGs exhibited rupture of the epididymal ducts with a focal aggregation of macrophages in the interstitium. Although some lesions revealed no unequivocal evidence of rupture of the epididymal ducts, we diagnosed them as SGs if they were further characterized by engorgement of epididymal ducts with spermatozoa and multinucleated spermatozoa in duct lumina. Lesions were found in the proximal caput of the epididymis and/or efferent ducts, but not in the corpus and cauda. In the testis, the incidences of germinal epithelial atrophy (GEA) were increased in male mice at 2 years in a dose-related manner (1/50, 4/50, 8/50, 12/50, respectively). All the mice with epididymal SG developed testicular GEA, but not vice versa. The grading score of GEA in the testis was more severe in mice with SGs than in those without. No correlation occurred between the presence or absence of the SGs and the distribution of atrophic seminiferous tubules. No epididymal SGs or testicular GEA were observed in interim-sacrificed mice. In the 2-year rat study, no differences occurred in mortalities between the control and treated groups. In the epididymis SGs were recognized only in 1 male rat in each treated group. Most of the rats in each group, including the control, displayed GEA of the testis, which may have resulted from the interstitial-cell proliferation. The results suggest that 2-MI can induce SGs of the epididymis in B6C3F1 mice, not F344/N rats, treated for 2 years and that the SGs may sometimes be associated with testicular GEA.
P31 Spontaneous Gastrointestinal Neoplasms in Control CD-1 Mice on a Two-Year Carcinogenicity StudyWyeth Research, Chazy, New York, United States Spontaneous gastrointestinal neoplasms are described infrequently in CD-1 mice on 2-year carcinogenicity studies. In this report, we describe the morphological appearance of a jejunal adenocarcinoma in a male CD-1 mouse, and an undifferentiated gastric carcinoma with hepatic metastases in a female CD-1 mouse. The male was electively euthanized on day 651 of the study, with clinical signs that included distended abdomen, pale appearance, and coolness to the touch. At necropsy, a 0.5 cm in diameter, firm mass was found in the jejunal wall. Microscopically, the submucosal mass was composed of dense sheets of deeply basophilic round to ovoid cells, frequently forming irregular acini that contained palely eosinophilic, necrotic debris. Nuclei were round, and generally contained a single nucleolus. Mitotic figures were seen at a frequency of 1–2/hpf (x40). A diagnosis of small intestinal adenocarcinoma was made. Metastatic lesions were not found, but the mouse did have concurrent hepatocellular adenoma and hepatic hemangiosarcoma. The female mouse was electively euthanized on day 585 of the study, with clinical signs that included rough hair coat, distended abdomen, pale appearance, and coolness to the touch. At necropsy, a 2.0 cm diameter, pale mass was visible through the gastric serosa. Histologically, the mass was composed of dense sheets of amphophilic to basophilic cells with indistinct cell borders. Rare acini were seen, and necrosis was common. Nuclei were variable in size, and generally contained a single nucleolus. Mitoses were uncommon. Hepatic metastasis was present. A diagnosis of undifferentiated gastric carcinoma was made. Although both of these tumors have been previously identified in CD-1 mice, it is important to expand the historical control database of such uncommon neoplasms. Such data are helpful in interpretation of tumor statistics, and potential test article related effects.
P32 Comparative Morphology and Incidence of Meningiomas in Rats and Mice from Studies of the National Toxicology ProgramNational Institute of Environmental Health Sciences, Research Triangle Park, North Carolina, United States To clarify the incidence and pathological characteristics of rodent meningiomas, specimens from rats (various strains), B6C3F1 mice, and p16-deficient mice from the National Toxicology Program (NTP) archives (1971–2003) were evaluated and classified. Society of Toxicologic Pathology (STP) and World Health Organization (WHO) morphological criteria were utilized to classify the lesions and many neoplasms were further characterized by histochemical methods (PAS, alcian blue, silver, and trichrome stains), immunohistochemical stains (vimentin, cytokeratin, and GFAP), and/or ultrastructural analysis. Meningiomas are rare tumors in rats in NTP studies: only 37 cases were found and they included F344, Marshall, and Osborne-Mendell strains. The overall incidence of meningioma in control rats was 4/4549 (0.09%) in males and 1/4585 (0.02%) in females. The remaining 32 meningiomas occurred in various chemical treatment groups, but were not clearly related to treatment. The neoplasms consisted of spindle, meningothelial, or primitive tumor cells, and were classified as either meningotheliomatous (19%), fibrous/fibroblastic (51%), transitional (14%), psammomatous (8%), or anaplastic (8%). The majority of tumors showed a nodular and compressive proliferation pattern and occasional neoplasms had extensive invasion. One male F344 rat had extracranial metastasis to the lung. Then, 164 rats with granular cell tumor of the meninges were found in the NTP archives and excluded from this study because they have been previously described and are of uncertain origin. In control B6C3F1 mice, the overall incidence of meningiomas was 0/4832 (0%) in males and 2/4928 (0.04%) in females. The remaining 23 meningiomas were from various treatment groups, however, none were clearly related to treatment. The neoplasms consisted of spindle, myxomatous or meningothelial tumor cells, and were classified as myxoid (44%), meningotheliomatous (28%), or fibrous/fibroblastic (28%) subtype. In p16-deficient mice (B6/129-Cdkn2atm1Rdp (N2)), meningiomas were induced in up to 50% (5/10) of the males after in utero exposure to ethylnitrosourea. The meningiomas of the p16-deficient mice were highly cellular and of the fibrous subtype. The majority of mouse neoplasms demonstrated spreading invasion along the meninges with compression and occasional invasion, primarily perivascular, of the brain parenchyma. Extracranial distant metastasis was not observed in the mouse. In summary, spontaneous rodent meningiomas in NTP studies are relatively rare, often invade adjacent tissue, and a sex predisposition was not found. Chemically induced meningiomas were frequent in the p16-deficient mouse and this may be a good animal model to study meningiomas.
P33 Alpha Glutathione S-Transferase as a Novel Biomarker for Monitoring Chronic Methotrexate Hepatotoxicity
1 Glasgow Hospitals
Methotrexate is a commonly used drug for treating severe psoriasis and other severe immunological diseases. It is a known hepatotoxin and it is recommended that liver function be monitored in patients and that liver biopsies are performed on patients after they have received fixed cumulative doses of 1–1.5 g of the drug. Improved monitoring of liver status could reduce the need for routine biopsies, saving costs and reducing patient morbidity while identifying those most at risk. Alpha glutathione S-transferase (
P34 Myeloproliferative Disorder in Rotenone Treated Tg.AC and FVB/N Mice: Histologic Spectrum
1 Department of Veterinary Pathobiology, College of Veterinary Medicine, University of Illinois, Illinois, Urbana, United States Myeloproliferative disorder (myeloid dysplasia) refers to a group of hematopoietic disorders characterized by proliferation of one or more of the hematopoietic cellular lineages resulting in leukemias of cell lineages other than lymphocytes. Myeloproliferative disorder was diagnosed in 24- to -37 weeks old, female mice: 15/28 Tg.AC mice and 8/23 FVB/N mice treated topically with rotenone for 26 weeks, 7/9 positive control Tg.AC mice treated with 12-O-tetradecanoyl-phorbol-13-acetate (TPA), but not in negative control Tg.AC or FVB/N mice treated with vehicle. Tissues from affected mice were evaluated for distribution of lesions and scored for lesion severity. Severity was scored from mild (1+), in which there were small, scattered, perivascularly oriented foci of the myeloid cell population, to severe (4+), in which the myeloid cells effaced normal architecture. The bone marrow was markedly hypercellular with areas in which blasts comprised greater than 20% of the cells. The maturation of the myeloid lineage did not follow through to completion. There was a left shift with presence of giant metamyelocytes. In the liver, the myeloid cells were most abundant in periportal areas with disruption and infiltration past the limiting plate. In severe cases, there was bridging across adjacent portal areas. Central veins contained foci of the myeloid cells beneath the subendothelium that bulged into the lumen. In the spleen, lymphoid follicles were depleted and the red pulp was multifocally replaced by the myeloid cell population. Other commonly affected organs were kidney, with the pelvis and parenchyma adjacent to large blood vessels most commonly affected, and lung, with parenchyma adjacent to large blood vessels affected. In severely affected mice, lymph node sinuses were expanded by large numbers of the myeloid cells. Eosinophilic crystals were observed frequently in the lung and liver of these mice (see Osborne et al, this meeting). The myeloid cells exhibited characteristics of neoplastic, inflammatory, and hematopoietic cells. Rotenone-treated mice allowed to recover for 6 weeks were not different than those without a recovery period. In summary, a myeloproliferative disorder is described in rotenone-treated Tg.AC and wild-type FVB/N mice as well as in TPA-treated Tg.AC mice.
P35 Development of Flow Cytometric Assays to Measure Immunomodulatory Effects of Environmental Contaminants on Oyster Cellular Immune FunctionsUniversity of Connecticut, Storrs, Connecticut, United States Though bivalve mollusks have been used extensively as ocean sentinel monitors for chemical contamination, little is known of the relationship between toxic exposure and bivalve biologic response. Of special interest in our laboratory are the effects on the immune system, which is among the most sensitive to the effects of pollutants, and is central to health. Nevertheless, assays were not available to precisely quantify immune functions in oysters. To address this, cellular immune mechanisms of the Eastern Oyster (Crassostrea virginica) were quantified at the single cell level utilizing flow cytometry. Phagocytosis was measured using fluorescent beads. Respiratory burst activity was quantified as the increase in dichlorofluorescein-associated fluorescence upon stimulation. Apoptosis was evaluated with the Annexin V assay. Subpopulations of hemocytes (granulocytes and hyalinocytes) were identified with unique functional characteristics. Granulocytes were most active at phagocytosis and peroxide production while hyalinocytes were relatively inactive. Annexin V assay application allowed quantification of hemocyte apoptosis with water salinity dependent changes. Flow cytometry can rapidly, accurately, and directly quantify the morphology and function of a large number of individual cells, and will lead to a better understanding of the bivalve immune system. Lipophilic organic contaminants, such as PCBs (polychlorinated biphenyls), are ubiquitous in aquatic ecosystems. They are highly resistant to degradation in marine environments and are known immunotoxicants in laboratory animals, reducing resistance to infectious diseases. The last 30 years have seen a marked rise in infectious diseases caused by protozoa in cultured oysters on the Atlantic and Gulf coasts. A better understanding of the immunomodulatory effects of these pollutants in oysters will further the understanding of aquatic contamination and its consequences.
P36 Assessment of Carbaryl-Induced Immunotoxicity in Chickens
1 The Royal Veterinary College, University of London, North Mymms, Herts, United Kingdom Carbaryl is a wide-spectrum carbamate insecticide that controls over 100 species of insects on citrus, fruit, cotton, forests, lawns, nuts, ornamentals, shade trees, and other crops, as well as on poultry, livestock, and pets. Acute toxic effects of carbaryl are well documented but its chronic effects in low dose on immune system are not well studied. The present study was planned to investigate the effect of the methyl carbamate pesticide carbaryl on macrophage functions, lymphocyte proliferation, and delayed type hypersensitivity response. One-day-old broiler chicks were procured and randomly divided in 2 groups. Group I served as control while group II birds were given carbaryl at 20 ppm (No observable effect level, NOEL) in feed for a period of 3 months. To measure the functional activity of phagocytic cells, Nitroblue Tetrazolium (NBT) reduction test was performed on peripheral blood leucocytes. Concanvalin A (Con-A) and lipopolysaccharide-stimulated proliferation of T and B lymphocytes was assessed using an MTT dye method. At the end of the experiment, phagocytic capacities of macrophages were significantly reduced in carbaryl treated group. Lymphocyte proliferation responses to Con-A and LPS were 23% and 28% lower in chickens fed with carbaryl. Delayed hypersensitivity reaction to tuberculin was reduced to 77% of control values indicating the inhibition of cell-mediated immune response. Data from present study is suggestive of an immunosuppressive effect of carbaryl in chickens.
P37 Concordance of Toxicogenomic Predictions and Mechanistic Analysis for Compounds Tested in Both Rat Liver and Primary Rat HepatocytesGene Logic Inc., Gaithersburg, Maryland, United States Even with an assortment of cell-based assays available today, early detection of compounds that are capable of inducing liver toxicity is still a significant problem in the toxicology field. A cell-based assay that was able to identify multiple types of liver toxicity and provide richer information regarding mechanism of toxicity would improve upon current technologies. Comparisons between toxicogenomic data derived from rat (liver) or primary rat hepatocytes were evaluated to determine the feasibility of using the combination of primary rat hepatoyctes and microarray analysis for detecting liver toxicity. Predictive models used in this study were developed based on gene expression information in the ToxExpress database that is derived from rat (liver) or primary rat hepatocytes analyzed with the Affymetrix RGU34 chip set following treatment with a large number of compounds. For the in vivo to in vitro comparisons, a concordance of 75% was observed for the compounds examined by their respective predictive models. Of the compounds examined, 15% were only identified by the liver predictive models while 10% of the compounds were only identified by the rat hepatocyte modeling system. Notably, the rat hepatocyte models were able to identify a pharmacologically diverse set of compounds capable of inducing steatosis and/or hepatitis such as Valproic acid (Antiepileptic), Amiodarone (Antiarrhythmic), Tetracycline (Antimicrobial) Diclofenac (NSAID), and Indomethacin (NSAID), agents that are difficult to detect by conventional techniques. In addition, both the rat and primary rat hepatocyte predictive models were able to detect clofibrate as a liver toxin. A comparative analysis of microarray data from the rat (liver) and rat hepatocytes indicated a very similar response in genes associated with beta oxidation such as RATACOA1, Mte1, CPT1a, CPT2, CROT, Ech1, Acat, and Acaa and peroxisome proliferation associated genes such as Pex11a, Hsd17b4, Decr2, and Abcd3 providing evidence that a similar biologic response occurred in both biological systems. These results provide evidence that primary rat hepatocyte system in conjunction with microarray analysis is a valuable in vitro assay tool for detecting liver toxins and it also provides precious mechanistic information regarding the observed toxicity.
P38 Histopathological Characterization of the Skeletal Myopathy in rasH2 Mice: A Comparative Study between RasH2 and mdx Mice
1 Mitsubishi Pharma Corporation, Kisarazu-shi, Chibaken, Japan Skeletal myopathy was found to be approximately 100% in the rasH2 mice. Microscopically, this shows features similar to muscular dystrophy. To confirm the detailed characteristics of skeletal myopathy in rasH2 mice, the muscles obtained from male rasH2 mice aged 10–13 weeks were investigated by using HE, Gomori trichrome, and NADH-TR stain, as well as immunohistochemical analysis of MyoD (myogenic cell marker), dystrophin, and sarcoglycan (a major component of dystrophin-glycoprotein complex). The mdx, which is a dystrophin-deficient model for the Duchenne-type muscular dystrophy in humans, were used for comparison with the histopathologic features of rasH2 mice. In addition, age-matched, wild-type animals for each rasH2 and mdx mouse were examined. The results were that primary muscle fiber degeneration/necrosis and its regeneration and variation of fiber size were seen in both the rasH2 and mdx mice, however, there were some differences between them. In the HE and Gomori trichrome stains, scattered degeneration/necrosis and regeneration of muscle fibers and mononuclear cell infiltrations around the degenerative muscle fibers were observed in the rasH2 mice. There was no myopathic grouping of regenerating muscle fibers. In the mdx mice, degeneration/necrosis of muscle fibers accompanied by phagocytic cell infiltration were more prominent than that of rasH2 mice, and mineralization was sometimes seen at the necrotic muscle fibers. Most of the muscle fibers had central nuclei, suggesting regenerating fibers in areas other than the degenerative or necrotic area. Myopathic grouping of regenerating muscle fibers was frequently seen in the mdx mice. In the NADH-TR stain, muscle fibers showing a disorganized intermyofibrillar network and necrotic change were clearly demonstrated in both the rasH2 and mdx mice. Both mice showed neither fiber type grouping that suggests denervation of muscle fibers in the NADH-TR stain nor specific morphological changes (such as the rod structure or tubular aggregation seen in some types of human myopathy) in the Gomori trichrome/NADH-TR stains. No abnormality was observed in each wild-type mouse in the preceding examinations. Immunohistochemically, MyoD positive cells suggesting active myogenic differentiation were not frequent in the rasH2 mice, and expression patterns of dystrophin and sarcoglycan (positive around the rim of muscle fibers) were comparable between the rasH2 and the wild-type mice. In the mdx mice, MyoD positive cells were frequently seen in the area of myopathic grouping of regenerating muscle fibers. Dystrophin and sarcoglycan expressions were absent and weak, respectively, in the mdx mice compared to those of the wild-type mice. These results indicated that myopathic changes in rasH2 mice are based on the degeneration and regeneration of muscle fibers without specific structural changes seen in some types of human myopathy. The progress of muscle fiber degeneration and regeneration in rasH2 mice was considered to be more gradual than those of mdx mice at this age. Abnormality of dystrophin and sarcoglycan was not considered to be involved in the pathogenesis of muscular degeneration in rasH2 mice. Therefore, the cause of muscular degeneration in the rasH2 mice was thought to be different from that of mdx mice.
P39 Incidences of Selected Lesions in Control Harlan Sprague-Dawley Rats from Two-Year Toxic Equivalency Studies Performed by the National Toxicology Program
1 EPL, Inc., Research Triangle Park, North Carolina, United States Historical control incidences are an important consideration when interpreting data from a National Toxicology Program (NTP) study. The NTP has a long history of using Fisher F344 rats and has compiled a large database of incidences of lesions seen in control animals. This database is often used when the incidence of a lesion in the concurrent control group is outside the historical control range, or to provide support for the significance, or lack thereof, of very uncommon lesions. However, the NTP does not have the breadth of data collected on Harlan Sprague–Dawley (SD) rats, a strain commonly used in commercial toxicologic studies. Recently, the NTP conducted a series of 7 chronic studies using female Harlan SD rats to evaluate the toxic and carcinogenic effects of long-term exposure to dioxin and dioxin-like compounds and their mixture. This poster summarizes the changes found in the control animals used in those 7 studies, and compares the incidences of these lesions to those seen in Fischer F344 rats used in 2-year studies by the NTP and fed the same diet (Haseman et al., 2003). The overall survival rate in Harlan SD rats was 41.5%, with a mean lifespan of 628.7 days compared to a survival rate of 74.2% and a mean lifespan of 704.7 days for control female Fisher F344 rats. The maximum mean weekly body weight was 387.3 grams for Harlan SD and 325.6 grams for Fischer F344 rats. The most commonly observed neoplasms in the control Harlan SD rats were mammary gland fibroadenoma (71%), tumors of the pars distalis of the pituitary (41%) and thyroid gland C-cell tumors (30%). Female Fischer F344 rats had incidences of 44% for mammary gland fibroadenomas, 34% for tumors of the pars distalis, and 16% for thyroid gland C-cell tumors. Fischer F344 rats had a 15% incidence of clitoral gland tumors while the Harlan SD rats had an incidence of <1%. In contrast to Fischer F344 rats, the Harlan SD rats had a high incidence of squamous metaplasia of the uterus (44.2%). This lesion was seen as early as 14 weeks, thus making this strain of rats less desirable for toxicity and carcinogenicity studies where the uterus is a suspected target. The Harlan SD rats had a very low incidence of mononuclear cell leukemia (0.5%) when compared with the rates seen in Fischer F344 rats (24%). Mononuclear cell leukemia can cause significant secondary effects, especially in the liver; therefore, the Harlan SD might be a better choice for chronic studies where the liver is a suspected target. Haseman JK, Ney E, Nyska A, Rao GN. Effect of Diet and Animal Care/Housing Protocols on Body Weight, Survival, Tumor Incidences, and Nephropathy Severity of F344 Rats in Chronic Studies. Toxicologic Pathology, 31:674–681, 2003.
P40 Acute and Chronic Toxicity of Gentamicin in RatsF. Hoffmann-La Roche Ltd., Basel, Switzerland The acute and chronic toxicity of gentamicin was assessed in adult male Wistar rats treated once or once daily for 7 days at 6.25 mg/kg, 25 mg/kg or 100 mg/kg subcutaneously (SC) and necropsy was performed 24h after the last treatment. Control animals were given normal saline SC. The main lesions were noted in the kidney cortex, affecting P1 and P2 segments of the proximal convoluted tubules (PCT) and consisting of acute degeneration, necrosis, and regeneration of the tubular epithelium. The straight part (P3 segment) of the PCT in the outer zone of the outer medulla was less affected. Scattered degeneration, single-cell necrosis, and regeneration of tubular epithelial cells were noted after a single treatment at 25 and 100 mg/kg and after 7 days treatment at 6.25 mg/kg/day gentamicin. Degenerate tubular epithelial cells were swollen and had hypereosinophilic cytoplasm with periodic acid-Schiff (PAS)-positive granules. More severe and widespread tubular degeneration, necrosis, and regeneration were seen in rats treated at 25 and 100 mg/kg/day for 7 days. Epithelial cells lining most of the proximal convoluted tubules in the cortex were swollen, had indistinct cell outlines, and had markedly disrupted hypereosinophilic cytoplasm with numerous large, intensely PAS-positive granules. Nuclei were pyknotic, karyorrhectic, or missing. Cell debris forming eosinophilic granular casts plugged tubular lumina of rats treated at 100 mg/kg/day. Lesser amounts of an amorphous proteinaceous material (hyaline casts) were present in other tubules. Occasional granular or hyaline casts were found within tubules in the medulla. The brush border of affected PCT was often attenuated or sloughed. A dose-related increase in incidence and severity of tubular dilation of mainly distal tubules in the cortex was also seen. Individual or small clusters of regenerative tubular epithelial cells interspersed among degenerate, necrotic or normal cells were present in all rats treated once at 25 and 100 mg/kg. Treatment at higher doses for 7 days resulted in relatively widespread epithelial regeneration affecting entire tubules, particularly at 100 mg/kg/day. Regenerative epithelial cells were lightly basophilic and enlarged. Nuclei had prominent 1 to multiple nucleoli and scattered mitotic figures were also seen. Immunohistochemical detection of proliferating cell nuclear antigen (PCNA) confirmed the degree of regeneration in gentamicin-treated rats. The present study confirms that the incidence and severity of lesions attributable to gentamicin are related to dose and duration of treatment. Tubular epithelial degeneration, necrosis, and regeneration were seen as early as 24 hour after a single dose of gentamicin at 25 mg/kg and were more severe with prolonged treatment at higher doses. There were no significant findings after a single dose of gentamicin at 6.25 mg/kg.
P41 A 13-Week Toxicity Study of 3-MCPD in Drinking Water in Mice
1 Korea Food and Drug Administration, National Institute of Toxicological Research, Seoul, South Korea The compound 3-monochloropropane-1, 2-diol (3-MCPD) is a food processing contaminant, which has been detected in a wide range of foods and ingredients; 3-MCPD produced by the acid hydrolysis of soybean products is suspected to cause cancer. Furthermore, 3-MCPD is known to decrease sperm number, motility, and fertility rate. To evaluate 13-week toxicity of 3-MCPD, B6C3F1 mice were fed with drinking water for 13 consecutive weeks. B6C3F1 mice were scheduled to treat with 3-MCPD at doses of 0, 5, 25, 100, 200, and 400 ppm dissolved in drinking water for 13 weeks. Groups of 10 male and female B6C3F1 mice were fed diets and water ad libitum. The weight gain and, water and food consumption were recorded weekly from start to termination. The animals were examined twice a day for changes in health or behavior. According to the NTP guidelines, the vaginal cytology evaluation for female and sperm motility test at termination were performed to evaluate the effect of reproductive toxicity of 3-MCPD. At the end of experiment, the animals were sacrificed and subjected to complete necropsy and histopathological examination. The liver, kidneys, spleen, heart, adrenals, testis, and ovary were weighed. Blood chemistry and haematological parameters were evaluated in blood samples taken at termination. The body weights of mice at the highest dose were significantly reduced after week 8 for males and week 9 for females (p < 05). The animals receiving drinking water containing 3-MCPD at a concentration of 0, 5, 25, 100, 200, and 400 ppm were equivalent to mean daily intakes of 0, 0.94, 4.60, 18.06, 37.05, and 76.75 mg/kg per day for males and 0, 0.83, 4.15, 15.77, 31.75, and 64.26 mg/kg per day for females, for 13 weeks. Relative kidney and adrenal gland weights of males and kidney , adrenal gland, and ovary weights of females at the highest dose was significantly increased (p < 0.05). The mean estrus cycle of females at the highest dose was significantly increased (p < 0.05). The sperm motility of males at the highest dose was significantly decreased (p < 0.05). Tubular degeneration of kidneys was found in all groups, and no significant difference was found between groups. Vacuolar degeneration in the brain stem was found in 200 and 400 ppm treatment groups of males significantly (p < 0.05). As a results of this study, the 3-MCPD should be viewed as toxic substance, with a maximum tolerance dose (MTD) could be concluded as 37.05 mg/kg per day for males and 31.75 mg/kg per day for females.
P42 Pathology of Bacillus Anthracis Lethal Toxin in BALB/cJ and C57BL/6J Mice
1 SAIC-Frederick, NCI at Frederick, Frederick, Maryland, United States Bacillus anthracis toxin consists of 3 polypeptides: edema factor (EF), lethal factor (LF), and protective antigen (PA). LF and EF are only toxic when combined with PA, the latter being required for binding and translocation of toxin into a cell. Lethal toxin (LT), composed of LF and PA, is the major virulence factor of anthrax and accounts for most of the clinical manifestations of the disease in animals. Previous studies found mouse strains vary in their sensitivity to LT based on the sensitivity of the mouse strain’s macrophages. Thus pathogenesis of LT was interpreted to stem from macrophage-dependent endotoxic shock. We studied LT toxicity in BALB/cJ and C57BL/6J mice; previously reported to be "sensitive" and "resistant," respectively, on the basis of macrophage sensitivity. Three mice from each strain were euthanized 6, 12, 18, 24, 36, and 48 hours after receiving (ip)100 µg of LT, PA alone, or PBS. BALB/cJ became terminally ill earlier and with higher frequency than C57BL/6J. Histopathologic evaluation revealed similar lesions in both strains, though of later onset in the C57BL/6J mice. Significant lesions included apoptosis to focally diffuse necrosis in the metaphyseal bone marrow, apoptosis of myelogenous cells in the splenic red pulp and centrilobular hepatocellular coagulative necrosis. Crisis was due to vascular collapse and extensive hypoxia without hallmarks of TNF-<-mediated shock. Mild to moderate pulmonary and/or peritoneal effusions, edema/congestion of distal limbs, circulatory neutrophilia, and thrombocytopenia occurred in a majority of the mice of both strains. There was no evidence of disseminated intravascular coagulation, hemorrhage, or renal dysfunction; however, hepatic dysfunction, hypoalbuminemia and vascular/oxygenation insufficiency were documented in this and related experiments. Of 50 cytokines analyzed, BALB/cJ mice showed rapid but transitory increases over a 6-to12-hour period in specific factors including KC, MCP-1/JE, IL-6, MIP-2, G-CSF, GM-CSF, Eotaxin, FasL and IL-1. No changes in TNF-<-occurred. The C57BL/6J mice did not mount a similar cytokine response. The factors were not induced in vitro by LT treatment of toxin-sensitive macrophages. These findings indicate that LT causes morbidity in mice through a noninflammatory, TNF-< and FasL-independent mechanism involving hypoxic tissue injury with no requirement for macrophage sensitivity to toxin. Furthermore, clinical investigation of human anthrax treatments, which currently focus on anticytokine (sepsis) therapies, may require reevaluation. Funded in part with federal funds from the National Cancer Institute under contract NO1-CO-12400 to SAIC Frederick.
P43 Purified Anthrax Edema Toxin in Mice Causes Multi-Organ Damage
1 National Institutes of Health, Bethesda, Maryland, United States Anthrax edema toxin is composed of 2 polypeptides, edema factor and protective antigen. Protective antigen is the receptor binding subunit required for translocation of the catalytic moiety edema factor, an adenyl cyclase, into cells. We examined the effects of edema toxin in mice. Female BALB/cJ mice (11–12 weeks) were injected intravenously with purified anthrax edema toxin (37.5 µg edema factor +37.5 µg protective antigen). Three mice per time point, plus appropriate controls were examined at 3, 12, 24, 36, 48, and 60 hours post-inoculation. Mice were bled for hematology and serum chemistry analyses, euthanized, and necropsied for routine light microscopic histopathologic evaluation. Edema toxin caused degeneration and necrosis in adrenal glands, gastrointestinal tract, bone, heart, kidneys, lymphoid tissue, bone marrow, reproductive tract, and submandibular salivary glands beginning at 12 hours. The adrenal zona fasciculata was diffusely necrotic at 12 hours, and at subsequent time points. The adrenal medulla appeared unaffected. At 12 hours, there was multifocal necrosis of gastric, enteric, and cecal mucosa with hemorrhage; this change was found in most mice at later time points. Crypt cells were not affected. Osteoblast and osteocyte necrosis was observed at 24 hours, in the metaphyseal trabeculae of the growth plate in long bones. By 48 and 60 hours, there was loss of the metaphyseal bone, resulting in discontinuity of the trabeculae, with no appreciable regeneration of osteoblasts. In the heart, scattered degenerate myofibers were found at 24 hours. Myocardial degeneration increased in severity at subsequent time points. Vacuolation of cortical tubular epithelium in the kidneys was found at 12 hours. By 48 and 60 hours, cortical tubules were multifocally necrotic. Lymphocytolysis occurred by 12 hours and progressed to lymphoid depletion in all lymphoid organs (thymus, lymph nodes, spleen, gut associated lymphoid tissue). Loss of hematopoietic cells in the bone marrow was found at 24 hours, continuing through 60 hours. Necrosis of luminal epithelium in the uterus and oviduct was found in most mice at 24 hours and subsequent time points. Acinar cells in the submandibular salivary gland were vacuolated at 24 hours and multifocally degenerate by 60 hours. Cytotoxicity due to anthrax toxin has been attributed largely to lethal toxin (lethal factor plus protective antigen). Few in vivo studies have been performed to examine the contribution of edema toxin to anthrax-induced morbidity and mortality. This study demonstrates that edema toxin causes significant organ damage and highlights the need to develop therapeutics against edema toxin for the treatment of anthrax.
P44 Safety of DT388 IL3, a Fusion Toxin Consisting of a Truncated Diphtheria Toxin (DT388) Linked to Human Interleukin 3 (IL3), in Cynomolgus MonkeysWake Forest University Health Sciences, Winston-Salem, North Carolina, United States We developed a fusion toxin, DT388IL3, consisting of the catalytic and translocation domains of diphtheria toxin (DT388) linked to interleukin 3 (IL3) for the treatment of patients with acute myeloid leukemia (AML). Our goal in this study was to determine the maximum tolerated dose (MTD) and dose-limiting toxicity (DLT) of DT388IL3 in cynomolgus monkeys (Macaca fasicularis), which possess cross-reactive IL3 receptors. In our previous study we administered up to 6 doses of DT388IL3 at 40 µg/kg, 60 µg/kg or 100 µg/kg every other day to 3 groups of young adult monkeys (1 female, 1 male per group). In 5 of 6 monkeys, results showed a dosedependent increase in malaise and anorexia but no reproducible abnormalities in serum chemistries or blood counts. There was no evidence of organ damage by blood tests or histopathology. The sixth monkey, a female in the 100 µg/kg dose group died of moderate to severe vasculitis of multiple tissues. Based on these findings, we repeated the study with the 100 µg/kg dose group and added a group that received 150 µg/kg, in an effort to define the MTD. Two groups of female monkeys (2 females per group) were treated with up to 6 slow intravenous infusions of DT388IL3 at 100 µg/kg or 150 µg/kg every other day. One additional female monkey was treated as a negative control. Monkeys in the 100 µg/kg dose group showed moderate malaise and anorexia but no reproducible abnormalities in blood counts or serum chemistries. Moderate elevations of liver enzymes were noted in the 150 µg/kg dose group in addition to severe malaise and anorexia (DLT). No significant findings were revealed at necropsy of either group. Histopathologic findings revealed evidence of cerebral edema and neuronal hypoxia, regenerative myeloid hyperplasia, and hepatic degeneration and regeneration in the 150 µg/kg dose group. Similar lesions in the brain and bone marrow were observed in the 100 µg/kg dose group. DT388IL3 plasma half-life was approximately 30 minutes with a peak concentration of approximately 2 µg/ml (30,000 pM). The IC50 for AML blasts in vitro was 6 pM. Collectively our results suggest that DT388IL3 can be tolerated at doses up to 100 µg/kg, which is markedly higher than previously reported for other diphtheria toxin fusion proteins, and should in principle allow for dose-escalation with minimal toxic side effects. Based on these findings a Phase I clinical trial has recently been initiated with DT388IL3 for the treatment of AML.
P45 N-methyl-N-nitrosourea-Induced Retinal Degeneration in Animals: Mechanisms and Interventions
1 Toxicology Research Laboratories, Fujisawa Pharmaceutical Co., Ltd., Osaka, Japan Retinitis pigmentosa (RP) is a human disease characterized by loss of photoreceptor cells leading to visual disturbance and eventually to blindness. Establishment of reliable animal models is essential for better understanding of this disease and development of approaches to therapeutic intervention. We report here success in suppression in the rat of N-methyl-N-nitrosourea (MNU)-induced apoptosis leading to retinal degeneration. Retinal damage induced by MNU was highly reproducible and involved photoreceptor-cell loss that culminated and was obvious in all animals at approximately 7 days after a single systemic administration of MNU to mice (60 mg/kg), rats (60–75 mg/kg), hamsters (90 mg/kg), shrews (65 mg/kg), and monkeys (40 mg/kg). Extensive investigations in the rat revealed that MNU-induced photoreceptor-cell loss was due to apoptosis with downmodulation of Bcl-2 protein, upregulation of Bax protein, and activation of caspase families. Therapeutic approaches to control MNU-induced photoreceptor-cell loss in rats were evaluated by caspase-3 inhibitor, nicotinamide (NA), and docosahexaenoic acid (DHA). In the cases of caspase-3 inhibitor and NA, retinal damage 7 days after MNU treatment (60 mg/kg) was evaluated by retinal thickness and the retinal-damage ratio (length of damaged retina:entire retinal length). In the case of DHA, retinal tissue samples were examined when rats developed MNU-induced mammary tumors with a diameter of more than 1 cm, or at the termination of the experiment (20 weeks after MNU 50 mg/kg treatment). The first strategy we used to attempt to suppress photoreceptor-cell loss was shutting down the apoptotic cascade; caspase-3 inhibitor (4,000 ng Ac-DEVD-CHO, injected intravitreally 0 and 10 hours after MNU) partially suppressed disease progression, especially in the peripheral retina. Next, we tested the ability of nicotinamide, a water-soluble B-group vitamin (25–1,000 mg/kg NAinjected subcutaneously), to effectuate damaged-DNA repair and/or act by other mechanisms, and we found reversal of damage. Finally, dietary supplementation of 9.5% DHA counteracted photoreceptor-cell loss. Although the triggering mechanisms of pathogenesis and the apoptotic cascade may differ between rats and humans, suppression of MNU-induced photoreceptor apoptosis in rats may provide therapeutic information for human RP control.
P46 Higher Doses of Mucochloric Acid Induces PARP Activation and Mitochondrial Dysfunction in XRCC1 Deficient CellsUniversity of North Carolina, Chapel Hill, North Carolina, United States Epidemiologic studies indicate that long-term consumption of chlorinated disinfected water is associated with increases in colorectal and urinary bladder cancer. Chlorinated hydroxyfuranones are by-products produced by the action of chlorine on humic acids in drinking water. Up to 60% of the mutagenicity of chlorinated drinking waters, as assessed in bacterial mutagenicity testing, was associated with the occurrence of the chlorinated hydroxyfuranones. Although most of this mutagenicity was attributable to the presence of mutagen X (MX), mucochloric acid (MCA) appears to be present in disinfected water at concentration levels comparable to those of MX. Also, the mutagenic potency of MCA in mammalian cells was comparable to that reported for MX. While there is no direct link between MCA and cancer, we have shown that MCA is capable of damaging cells in culture. Our goal was to clarify whether the formation of single strand breaks (SSBs) was a mechanism by which MCA induces genotoxicity. We used the comet assay to detect SSB formation and a novel colorimetric assay that indirectly monitors poly(ADP-ribose) polymerase (PARP) activation during SSB repair. While using NAD+ as a substrate PARP binds to DNA nicks and recruits essential repair proteins to the damaged site. PARP hyperactivation depletes intracellular NAD+, and in doing so also decreases ATP, NADH, and NAD(P)H stores. To understand the role of PARP activation in depleting intracellular NAD(P)H we applied PARP inhibitors to our test systems. Furthermore, to discern whether the reduction in NAD(P)H was due to PARP activation or mitochondrial dysfunction, we monitored mitochondrial transmembrane potential after exposure to MCA, using confocal microscopy. We selected genetically matched Chinese hamster ovary (CHO) cells with and without functional XRCC1 (X-ray cross complementing group 1) proteins for exposure. XRCC1 is critical for genomic stability by coordinating SSB repair. In both assays we detected a significant increase in SSBs across all doses after exposure to MCA. Transfecting XRCC1 deficient CHO cells with hXRCC1 showed responses similar to proficient cells, supporting the role of XRCC1 in SSB repair after MCA exposure. In the comet assay, preexposure with the PARP inhibitor 1,5-dihydroxyisoquinoline (DHQ) induced equal or greater levels of DNA damage across all doses. While in the colorimetric assay, DHQ protected completely against a reduction in NAD(P)H at lower doses but was less effective at higher doses of MCA. We hypothesized that at higher doses of MCA the reduction in NAD(P)H was only partially due to PARP activation and that mitochondrial dysfunction also played a role. Confocal microscopy with tetramethylrhodamine methyl (TMRM), a mitochondrial membrane potential indicator, revealed that mitochondrial depolarization occurred only at high doses of MCA after 4 hours of exposure. These results explained the data obtained in colorimetric assay. We concluded that MCA exposure is not only associated with DNA strand break formation but also with mitochondrial dysfunction at higher doses of MCA. To our knowledge this is the first report associating exposure to water disinfection by-products with mitochondrial dysfunction.
P47 The Effects of Fumonisin B1 and Tumor Necrosis Factor-
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) is presumed to represent progression. Rats were injected with an initiating dose of diethylnitrosamine (DEN, 200 mg/kg IP) and received a partial hepatectomy 3 weeks after DEN injection. Gavage with corn oil or 10 µg/kg PCB126 in corn oil was started at 2 weeks after DEN administration. Sodium metaarsenite (75 ppm) in drinking water was started in 2 groups at 2 weeks and in 2 groups at 8 weeks after DEN. Rats were sacrificed at 8, 16, and 24 weeks. Arsenic as a single agent did not increase GST-P positive foci over DEN controls. There was an increase in GST-P positive preneoplastic foci in livers of rats exposed to PCB126 alone and in combination with arsenic. Previous work in our laboratory on arsenic as a promoter demonstrated inhibition of preneoplastic foci at 8 weeks. The addition of arsenic at 2 and 8 weeks post-DEN to animals with PCB exposure reduced the relative area of GST-P positive foci at 16 weeks, compared to PCB alone. By 24 weeks, this inhibitory effect was absent. Approximately 40% of large GST-P positive foci in the PCB- and PCB/arsenic-treated groups also stained for TGF
L; laboratory range 2.5–7.8 x 103/ 
GST) is a more sensitive indicator of acute hepatocyte injury than transaminases, but it has not previously been studied as an indicator of chronic toxicity. Thus, 44 subjects receiving low-dose methotrexate for psoriasis were monitored using alpha GST, ALT, AST, GGT, ALP and serum albumin. 
on the Accumulation of Sphingosine and Sphinganine in H9C2(2-1) and HepG2 Cells |
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- Nyska, A, Cumming, CA, Vainshtein, ANadler J, Ezov, N, Grunfeld, Y, Gileadi, O, & Behar, V. (2004). Electron microscopy of wet tissues: new imaging capabilities for renal pathology. Toxicol Pathol, 32, 3, in press. in press. in press.
[Abstract/Free Full Text] - Thiberge, S, Nechushtan, A, Sprinzak, D, Gileadi, O, Behar, V, Zik, O, Chowers, Y, Michaeli, S, Schlessinger, J, & Moses, E. (2004). Scanning electron microscopy of cells and tissues under fully hydrated conditions. Proc Natl Acad Sci USA, 101, 3346-51
[Abstract/Free Full Text]
P60 Scanning Electron Microscopy (SEM) Under Fully Hydrated Conditions: V alidation in Model of Hyaline-Droplet Nephropathy
1 Quantomix, Nes-Ziona Israel
2 Harlan Biotech, Ness Ziona, Israel
3 Quantomix, Rehovot, Israel
4 PAI, Durham, North Carolina, United States
5 National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina, United States
The capability to perform wet scanning electron microscopy (SEM) by introducing wet biological specimens into a scanning electron microscope was recently demonstrated to offer major advantages, such as the ability to probe into cells and tissues allowing clear observation of intra- and extracellular features; simple and rapid sample preparation; and the greater resolution of electron microscopy along with its ease of use and benefit of zoom. Samples are placed in sealed capsules and imaged through a resilient, electron-transparent membrane with a backscattered electron detector. The thin membrane, appropriately transparent, isolates the wet tissue from the vacuum. The contrast obtained may rely on the natural features of the imaged samples or can be enhanced by electrondense stains, like osmium to show lipid components, or uranyl acetate for other cellular components. The sample, several-mm thick, is prepared and imaged without embedding or sectioning and therefore suitable for rapid analysis. We applied this technique to D-limonene-induced nephropathy in which the accumulation of hyaline protein droplets is induced in proximal and distal convoluted tubules of the male rat kidney. We compared the wet SEM technology to conventional light microscopic histopathology using hematoxylin and eosin and Mallory-Heidenhain staining. Results indicated that proximal and distal convoluted tubules were simple to differentiate. Likewise, individual organelles, such as mitochondria, tubular epithelial nuclei, the brush border of the proximal convoluted tubule, and hyaline protein droplets were easily detected. Most often round, but occasionally variable in size and shape, intensely bright, white electrondense deposits were clearly defined within the cytoplasm of the epithelial cells lining individual tubules. These particular deposits were brightly eosinophilic following MH staining. The images obtained by wet SEM of the wet kidney specimens exhibited superior resolution, contrast, and magnification compared with those obtained by conventional light microscopy of paraffin sections. These advantages of the wet SEM technology indicate its potential utility in a wide range of applications in histopathology and toxicology.
P61 Fourier Transform Infrared Microscopy (FTIRM) for Identification of Crystal Deposits in Formalin Fixed Paraffin Embedded Tissue Sections
GlaxoSmithKline, Research Triangle Park, North Carolina, United States
The presence of crystal deposits in tissue sections is occasionally observed in certain natural diseases such as gout but can also be secondary to drug or chemical toxicity such as ethylene glycol toxicity. Precise in situ identification of crystals is useful for understanding the etiology or the mechanism of the disorder. In this report we describe the use of FTIRM to precisely identify crystals noted in tissue sections observed in 2 preclinical toxicity studies. In the first instance, the drug substance (compound A) was administered to rats as a part of the preclinical toxicity program. Routine histological examination revealed pale basophilic, lamellated to spicular crystalline material within a few renal tubules. These crystals did not stain with either PAS or with a silver stain (GMS) and were birefringent only in slides without a coverslip. In the second instance, the drug substance (compound B) was administered to rats and routine histological examination revealed intensely birefringent angular crystals in the liver, duodenum, jejunum and mesenteric lymph node. Since both the compounds were prodrugs that were converted to the active moiety in vivo it was not known whether the crystals were the prodrug, the active moiety, or a metabolite. To precisely identify the crystals observed in tissue sections, FTIRM was first used to obtain unique spectral chemical fingerprints using pure samples of the prodrug and the active moiety. Subsequently, stained (H&E) or unstained deparaffinized sections of the tissue section without a coverslip were used for characterizing the crystals. Areas of the tissue sections containing crystals were first located using brightfield or polarized microscopy, and infrared spectra of these foci were collected using a JY Horiba Labram HR Raman spectrometer that is fitted with a SensIR FTIR spectrometer. The spectra of the crystals within tissue sections were nearly identical to the spectra obtained with pure samples of the active moiety in both the cases. FTIRM is a rapid, nondestructive, in situ method for identification of crystalline material in formalin-fixed, paraffin-embedded tissue sections.
Toxicologic Pathology, Vol. 33, No. 1,
185-203 (2005)
DOI: 10.1080/01926230590881899
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