bluuebunny/crossref_metadata_2025 huggingface dataset:
Peer Review in Toxicologic Pathology
Authors: Ward Jerrold M. , Hardisty Jerry F. , Hailey James R. , Streett C. Spencer
Published: April 2009

Peer review of histopathology findings in safety assessment studies involving rodents and other animals is a relatively recent procedure in toxicologic pathology. It serves to ensure the integrity of the pathology evaluation in safety studies, encourages consistency of diagnostic criteria and use of common terminology, and provides a method of continuing education for participants. The use of a standardized system of pathology nomenclature and diagnostic criteria, such as the Society of Toxicologic Pathologist's Guides for Toxicologic Pathology, is of great value in the procedure. Pathology reviews may involve government-sponsored bioassay programs, in-house industrial corporations, or individual peer reviews suggested or required by government regulatory agencies. Pathology Working Groups can be an integral part of the review process. The extent of the peer review is primarily dependent on the study results; however, other variables such as confidence of the data, study size and duration, complexity, and purpose are also important considerations. Essential components of any peer review, however, include selection of tissues/lesions for review, by a reviewing pathologist, discrepancy resolution, data modification, and documentation of all aspects of the review process. Specific procedures for pathology peer review are discussed. Disagreements among pathologists discovered in peer reviews can be resolved by several methods and examples will be presented. The entire pathology peer review process should be a learning experience for all involved and can help ensure the integrity of animal toxicology studies used for important regulatory decisions involving the use of chemicals in our society.
Cause-of-Death Assignment at the National Center for Toxicological Research
Authors: Kodell Ralph L. , Blackwell Boon-Nam , Bucci Thomas J. , Greenman David L.
Published: April 2009

The system for assigning cause of death in animal studies of carcinogenicity at the National Center for Toxicological Research (NCTR) is described. An empirical study of the NCTR's experience with its current cause-of-death assignment system based on selected representative experiments is reported. Issues investigated include the degree of confidence associated with histologic cause-of-death assignment, potential age-, dose-, and sex-related differences in assigned grades of certainty of cause of death, and frequencies of identification of various organ-specific and systemic diagnoses as the cause of death. Implications for age-adjusted statistical tests of carcinogenicity that require cause-of-death data are discussed.
Enhanced Hexachloro-1:3-butadiene Nephrotoxicity in Rats with a Preexisting Adriamycin-Induced Nephrotic Syndrome
Authors: Kirby Gordon M. , Bach Peter H.
Published: April 2009

Renal damage was assessed by histopathology and urinalysis in male Wistar rats treated with either hexachloro-1:3-butadiene (HCBD; a single 170-mg/kg ip dose that caused proximal tubule necrosis), adriamycin (ADR; a single 5-mg/kg ip dose that caused minimal glomerular changes up to 35 days), or HCBD given 2 wk after ADR and compared with age-matched control rats for 21 days. Urinalysis values in ADR-treated rats showed minimal renal changes. HCBD significantly elevated urine volume (10-fold), protein (5-fold), glucose (175-fold), and brush border enzymes (10-600-fold), indicating severe proximal tubular damage, but most parameters returned to pretreatment levels 6 days after treatment. In ADR-pretreated rats subsequently given HCBD, both the urinary alkaline phosphatase and the ratio of kidney : body weight were significantly higher for longer periods. Histopathology demonstrated that the HCBD-induced proximal tubular lesion was confined to the outer stripe of the outer medulla. Advanced regeneration and repair was evident 21 days after HCBD treatment. In the ADR-pretreated rats the HCBD-induced lesion was more severe and affected the entire cortex and was characterized by marked tubular epithelial calcification, with little evidence of repair and tubular restitution 21 days after treatment. Enzyme histochemistry showed γ-glutamyltranspeptidase localized to the proximal tubules. After HCBD treatment the enzyme staining was lost and subsequently returned in parallel with histological recovery up to 21 days. The distribution and intensity of γ- glutamyltranspeptidase was unchanged in ADR-treated rats. The distribution and intensity of γ-glutamyltranspeptidase in kidneys of ADR-pretreated rats given HCBD had not returned to normal by day 21. The results of this study indicate that pretreatment with ADR increases HCBD-induced nephrotoxic damage and decreases renal cortical repair capacity.
Carcinogenicity of Dietary Dimethylnitrosomorpholine, N-methyl-N'-Nitro-N-Nitrosoguanidine, and Dibromoethane in Rainbow Trout
Authors: Hendricks Jerry D. , Shelton Dennis W. , Loveland Patricia M. , Pereira Cliff B. , Bailey George S.
Published: April 2009

Eighteen-mo feeding trials of rainbow trout were used to test the carcinogenicity of 5 chemicals in this species. A single exposure level was used for each substance. The doses and chemicals tested were 1,556 ppm 2,6-dimethylnltrosomorpholine (DMNM), 500 ppm N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), 2,000 ppm 1,2-dibromoethane (DBE), 2,000 ppm 1,1-dichloroethylene (DCE), and 200 ppm cyclophosphamide (CP). Liver and/or glandular stomach neoplasms were produced by DMNM (liver and stomach), MNNG (stomach), and DBE (chiefly, stomach tumors). In addition, DMNM produced a low incidence of swimbladder papillomas and caused testicular atrophy in 50% of treated males. DCE and CP produced no neoplasms at the exposure levels used. No evidence of other chronic toxicity was seen for any of the 5 compounds.
Sequential Appearance and Ultrastructure of Amphophilic Cell Foci, Adenomas, and Carcinomas in the Liver of Male and Female Rats Treated with Dehydroepiandrosterone
Authors: Metzger Christel , Mayer Doris , Hoffmann Herbert , Bocker Thomas , Hobe Gerhard , Benner Axel , Bannasch Peter
Published: April 2009

Dehydroepiandrosterone (DHEA), a hormone of the adrenal cortex, acts as a peroxisome proliferator and hepatocarcinogen in rats upon long-term treatment with high doses in the diet. The aim of the present study was to identify the site of origin of hepatocellular neoplasms and the sequence of preneoplastic lesions. Twenty-five female and 25 male rats were given 0.6% DHEA in the diet; 25 animals of each sex were controls. Groups of 5 treated and untreated animals were sacrificed after 4, 20, 32, 70, and 84 wk. Amphophilic cell foci were detected after 32 wk of treatment; they developed from the liver parenchyma almost exclusively in the vicinity of portal tracts. Adenomas of the amphophilic or amphophilic/tigroid cell phenotype were observed at 70 wk of treatment. Highly differentiated hepatocellular carcinomas presenting a similar cellular phenotype occurred after 70-84 wk. The incidence of hepatocellular carcinomas was 44% in female and 11% in male rats. Ultrastructural studies of the amphophilic cell foci and tumors revealed a marked proliferation of mitochondria and a moderate proliferation of peroxisomes in all lesions. In addition, a very strong peroxisome proliferation was observed in perivenular hepatocytes in the liver of female rats. Peroxisomes usually lacked core and showed flocculent matrices. In male rats, weak peroxisomal proliferation was observed. Typical morphological abnormalities of these peroxisomes were paracrystalline inclusions of striated appearance. Although the most prominent peroxisome proliferation was observed in perivenular hepatocytes, these cells did not seem to be involved in tumor development. In contrast, the morphological similarity of the amphophilic cell foci and the amphophilic/tigroid cell adenomas and carcinomas, their coincident localization near portal tracts, and the sequential appearance of these lesions suggest that the amphophilic cell foci represent an early stage in DHEA-induced hepatocellular neoplasia. Mitochondrial proliferation as the most prominent feature in all stages of this model of hepatocarcinogenesis may offer a new approach for analysis of hepatocarcinogenesis induced by DHEA and possibly other peroxisomal proliferators.
Phytic Acid, an Iron Chelator, Attenuates Pulmonary Inflammation and Fibrosis in Rats after Intratracheal Instillation of Asbestos
Authors: Kamp David W. , Israbian Vatche A. , Yeldandi Anjana V. , Panos Ralph J. , Graceffa Philip , Weitzman Sigmund A.
Published: April 2009

Reactive oxygen species, especially iron-catalyzed hydroxyl radicals (·OH), are implicated in the pathogenesis of asbestos-induced pulmonary toxicity. We previously demonstrated that phytic acid, an iron chelator, reduces amosite asbestos-induced OH generation, DNA strand break formation, and injury to cultured pulmonary epithelial cells (268 [1995, Am. J. Physiol. (Lung Cell. Mol. Physiol.) 12: L471-480]). To determine whether phytic acid diminishes pulmonary inflammation and fibrosis in rats after a single intratracheal (it) instillation of amosite asbestos, Sprague-Dawley rats were given either saline (1 ml), amosite asbestos (5 mg; 1 ml saline), or amosite treated with phytic acid (500 μM) for 24 hr and then instilled. At various times after asbestos exposure, the rats were euthanized and the lungs were lavaged and examined histologically. A fibrosis score was determined from trichrome-stained specimens. As compared to controls, asbestos elicited a significant pulmonary inflammatory response, as evidenced by an increase (∼ 2-fold) in bronchoalveolar lavage (BAL) cell counts at 1 wk and the percentage of BAL neutrophils (PMNs) and giant cells at 2 wk (0.1 vs 6.5% and 1.3 vs 6.1%, respectively; p < 0.05). Asbestos significantly increased the fibrosis score at 2 wk (0 ± 0 vs 5 ± 1; p < 0.05). The inflammatory and fibrotic changes were, as expected, observed in the respiratory bronchioles and terminal alveolar duct bifurcations. The increased percentage of BAL PMNs and giant cells persisted at 4 wk, as did the fibrotic changes. Compared to asbestos alone, phytic acid-treated asbestos elicited significantly less BAL PMNs (6.5 vs 1.0%; p < 0.05) and giant cells (6.1 vs 0.2%; p < 0.05) and caused significantly less fibrosis (5 vs 0.8; p < 0.05) 2 wk after exposure. We conclude that asbestos causes pulmonary inflammation and fibrosis in rats after it instillation and that phytic acid reduces these effects. These data support the role of iron-catalyzed free radicals in causing pulmonary toxicity from asbestos in vivo.
The Riddle of Hepatic Neoplasia in Brown Bullheads from Relatively Unpolluted Waters in New York State
Authors: Spitsbergen Jan M. , Wolfe Marilyn J.
Published: April 2009

Since 1985, pathologists at Cornell University have investigated the causes of lesions in freshwater fish throughout New York waters in order to clarify possible impairment of fish health by environmental contaminants. Fishermen and biologists alerted us to several relatively protected reservoirs and ponds in which we have found no evidence of elevated levels of anthropogenic environmental contaminants but in which up to 100% of brown bullheads exhibited skin neoplasia. Complete necropsies and histologic study revealed that over 30% of mature brown bullheads from some of these sites had benign or malignant hepatocellular or biliary liver neoplasia. Up to 50% of brown bullheads had benign or malignant liver neoplasia in other relatively unpolluted waters with no evidence of skin neoplasia in bullheads. Multiple samplings of brown bullheads from several of these sites have revealed puzzling variability in the prevalence of skin, liver, and other neoplasia in these fish populations. The cause of these striking epizootics of neoplasia in brown bullheads in unpolluted waters in New York State remains unclear. We hypothesize that natural carcinogens such as N-nitroso compounds formed in aquatic sediments or radon from geologic formations may contribute to epizootic fish neoplasia in New York waters.
Hepatocyte Clusters in the Spleen: A Normal Feature of Some Populations of Brown Bullheads in New York State
Authors: Spitsbergen Jan M. , Wolfe Marilyn J.
Published: April 2009

Since 1985, fish pathologists at Cornell University have studied lesions in freshwater fish inhabiting natural waters throughout New York State in order to clarify possible adverse effects of contaminants on fish health. In the course of these studies, we have conducted complete necropsies on over 400 brown bullheads and histologic examinations of the major organs of over 370 of these fish. In the course of our histologic studies, we observed islands of well-differentiated hepatocytes in the spleens of neoplasm-free brown bullheads from 2 of the 37 diverse lakes, rivers, and ponds that we sampled. The splenic hepatocyte islands occurred in 11-100% of fish from the 2 affected sites, with 1-50 hepatocyte clusters per section of spleen. Hepatocyte islands measured up to 1 mm in diameter. Unlike the metastases of hepatocellular carcinomas, which we have described elsewhere in this issue (9), these islands of hepatocytes in the spleens of neoplasm-free fish were not evident grossly. These hepatocyte clusters in the spleen of certain populations of brown bullheads may lead to confusion in studies of neoplasia in brown bullheads. Caution is advised in interpretation of metastasis of liver neoplasms to the spleen of brown bullheads.
The Use of Immunohistochemistry for Evaluating the Liver
Authors: Hall William C. , Rojko Jennifer L.
Published: April 2009

Immunohistochemistry has been utilized in recent years primarily for diagnosis of infectious diseases of the liver, especially in humans. The utility of immunohistochemistry has extended to experimental and toxicologic pathology in a variety of areas: identification of cell phenotype, cell receptors, cytokine and chemikine production, and functional cell changes such as enzyme induction. In addition, markers for experimental carcinogenesis studies are detectable by immunohistochemical changes as well as novel antigen induction such as placental glutathione-S-transferase, oncofetal proteins, oncogene products, and typing of neoplasms. Immunohistochemistry is also used to detect the origin and function of various cell types in developmental and toxicity studies. Careful use of immunohistochemical procedures in conjunction with routine pathology and molecular techniques enhance the ability of the toxicologic pathologist to diagnose unique conditions and to understand mechanisms of lesion development.
Emerging Issues in Mouse Liver Carcinogenesis
Authors: Ward Jerrold M. , Shibata Masa-Aki , Devor Deborah E.
Published: April 2009

The mouse liver is the primary target site for carcinogenesis of more than 200 chemicals (including pesticides. food additives, pharmaceuticals, and industrial intermediates) tested in long-term toxicity safety assessment assays. Mouse liver tumors develop through defined morphological stages (similar to those found in other species) whether their origin is of undetermined etiology (spontaneous) or induced by chemicals. The morphologic type of hepatocytes in the various stages of hepatocarcinogenesis is sometimes associated with the specific inducing agent. Liver tumors developing in toxic livers often have more benign appearances and may progress to carcinomas at a slower rate than tumors developing in histologically normal livers. Specific tumors, dependent on the inducing chemical, may regress under defined protocols. Genotoxic and nongenotoxic mouse hepatocarcinogens each may induce tumors of either high malignant or low malignant potential. Liver tumors with specific H-ras oncogene mutations may appear morphologically and biologically similar to those without proven ras mutations. Thus, distinguishing mechanism of carcinogenesis by liver tumor morphology and mutation spectra may be difficult. Additionally, the presence of liver tumors with a morphology and a ras oncogene mutation spectrum characteristic of spontaneous tumors in histologically normal livers of mice exposed to a nongenotoxic test chemical may indicate promotion of spontaneous hepatocarcinogenesis by one of several potential mechanisms. Further research into the mechanisms responsible for the increased incidences of liver tumors in mice exposed to test chemicals could enhance human cancer risk assessments.
Hepatotumorigenicity and Peroxisome Proliferation Induced by the Hypolipidemic CI-924 in a Two-Year Study in Male and Female B6C3F1 Mice
Authors: Walker Robin M. , Wojcinski Zbigniew W. , Hofstra Angela H. , King Lena M. , Rogers Joanne E. , Baker Kenneth W. , Chang Pil-Kyu , Smith Graham S.
Published: April 2009

The hepatic tumorigenicity of CI-924 (5,5'-(1,1'-biphenyl)-2,5-diylbis(oxy)(2,2-dimethylpentanoic acid)), a hypolipidemic agent, was evaluated in 50 B6C3F1 mice/sex/dose given drug in the diet at 0, 5, 25, and 75 mg/kg/day for 2 yr. Peroxisomal and drug-metabolizing enzyme determinations, as well as ultrastructural evaluations, were conducted in subsets of these same groups, because drugs of this class cause peroxisome proliferation and hepatic tumors in rodents. CI-924 elicited dose-dependent increases in the incidence of hepatocellular adenomas and carcinomas in both sexes that were statistically significant at 75 mg/kg. Stereologic evaluation revealed significant increases in hepatocellular peroxisome volume ratio, due to increased numbers of peroxisomes, in females at all doses and males at 75 mg/kg. Peroxisomal enzyme activity measurements revealed no change in catalase, but dose-dependent increases in carnitine acetyltransferase and cyanide-insensitive β-oxidation in both sexes. Peroxisome proliferation, determined biochemically or ultrastructurally, was twice as great in females compared to males. Total cytochrome P-450 was increased in both sexes given 75 mg/kg. There were dose-dependent decreases in glutathione S-transferase in males and increased glutathione peroxidase in both sexes at 25 and 75 mg/kg. In conclusion, this study demonstrated that while CI-924 induced hepatic tumors in male and female B6C3F1 mice the associated peroxisome proliferation, while moderate in females, was only weak in the males after 2 yr of exposure.
Increased Rate of Apoptosis Correlates with Hepatocellular Proliferation in Fischer-344 Rats Following Long-Term Exposure to a Mixture of Groundwater Contaminants
Authors: Constan Alexander A. , Benjamin Stephen A. , Tessari John D. , Baker Dale C. , Yang Raymond S.H.
Published: April 2009

Apoptosis was evaluated in the livers of Fischer-344 rats following observations of increased hepatocellular proliferation from exposures, at low parts per million (ppm) levels, to a drinking water mixture of 7 groundwater contaminants during a 6-mo time-course study. The 7 chemicals used are among the most frequently detected contaminants associated with hazardous waste sites: arsenic, benzene, chloroform, chromium, lead, phenol, and trichloroethylene. Significant increases in 5-bromo-2'-deoxyuridine hepatocellular labeling were present in a unique pattern surrounding large hepatic veins (0.5-2.0 mm). This did not appear to be a regenerative response due to cytotoxicity, as assessed by the absence of increased plasma enzyme activity and the absence of hepatocellular lesions. Immunohistochemical staining for apoptosis, using the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) method showed patterns of labeling in treated animals that directly correlated to areas of increased hepatocyte proliferation. Apoptotic activity was maximum at the 1-mo exposure time point, whereas proliferating hepatocytes reached a maximum rate at the 10-day time point. This may have been triggered as a compensatory response to the increased cell proliferation or as a protective response to remove cells with altered DNA due to chemical mixture exposure. The principal findings of this paper are that (a) apoptosis directly correlated with changes in cell proliferation; (b) observed effects were produced by repeated exposures to a relatively low-level chemical mixture; and (c) the TUNEL method detected apoptotic cells at very early and late stages, potentially increasing the observable time period for apoptosis.
Reactivity of Tissue-Specific Antigens in N-Methyl-N'-Nitro-N-Nitrosoguanidine-Induced Neoplasms and Normal Tissues from Medaka (Oryzias latipes)*1
Authors: Bunton T.E. , Wolfe Marilyn J.
Published: April 2009

To further characterize the distribution of tissue-specific antigens in fish neoplasms, juvenile medaka were exposed to 30 mg/L of N-methyl- N'-nitro- N-nitrosoguanidine (MNNG) for 1 hr and allowed to grow out for up to 16 mo. Using a streptavidin peroxidase technique, keratin, vimentin, and neurofilament intermediate filament proteins, and actin and S-100 proteins were labeled in MNNGinduced neoplasms and normal medaka tissues using specific monoclonal or polyclonal antibodies. In vascular tumors, rhabdomyosarcoma, and teratoma, muscle tissues were positive for actin. Other sarcomas including hemangiopericytoma, fascial sarcoma, and undifferentiated sarcoma were negative for all antibodies tested. An unusual scale-associated neoplasm, composed of clusters of scale-forming cells surrounding spicules of scale, had keratin-positive stroma. The epithelial neoplasms were also positive for keratin, except for pancreatic acinar carcinoma, which had limited positivity. Both teratoma and olfactory carcinoma had S-100-positive intraepithelial cells morphologically reminiscent of neurosensory epithelial cells, which were S-100 positive in normal tissues. Although positive reactivity in fish tissues correlated with mammalian data, the antibodies used were raised against mammalian antigens. Therefore, a negative reation may be indicative of lack of antibody sensitivity to specific fish antigens rather than absence of the antigen in the tissues. However, these data show that tissue-specific antigen detection may assist in elucidating the biology of neoplasia in fish.
Mesenteric Arteriopathy in the Rat Induced by Phosphodiesterase III Inhibitors: An Investigation of Morphological, Ultrastructural, and Hemodynamic Changes
Authors: Joseph E.C. , Rees J.A. , Dayan A.D.
Published: April 2009

A reproducible model of a phosphodiesterase III (PDE III) inhibitor-induced arteriopathy has been developed in the rat after subcutaneous administration of SK&F 95654. Administration of this potent PDE III inhibitor induced an arteriopathy of mesenteric arteries within 24 hr that was dose-related in intensity and incidence over the range 0.174, 0.348, 0.523, and 0.697 mmol/kg. The arteriopathy was restricted to muscular arteries of external diameter of 100-800 μm and was shown microscopically to be focal or segmental medial necrosis and hemorrhage. A time-course experiment, conducted from 3 to 24 hr postdosing, showed that the first changes observed 6 hr postdosing were on the endothelium followed by focal hemorrhages into the media at 12 hr postdosing, causing compression, degeneration, and necrosis of myocytes. From 16 hr postdosing, there was focal endothelial cell necrosis and loss of confluence. Leukocytes and activated platelets were found adhering to exposed basement lamina and seen to pass through endothelial gaps into the subintima. By 24 hr postdosing, medial necrosis was extensive with large areas of media replaced by erythrocytes, cell debris, and a few leukocytes and platelets.

The effect of 3 structurally dissimilar PDE III inhibitors administered subcutaneously at a dose of 0.697 mmol/kg was compared with that of SK&F 95654. The arteriopathy induced by these compounds were identical to that produced by SK&F 95654 with the incidence and severity of lesions ranked in the following order: SK&F 95654 > WIN 62582 > SK&F 94836, with no macroscopic lesions observed for SK&F 94120. Systolic blood pressure was measured for these 4 PDE III inhibitors at regular intervals over the 24-hr period postadministration by a plesthymographic method. The severity of the arterial lesions correlated with the magnitude of hypotension induced by these agents. It is postulated that the arterial damage is a consequence of profound vasodilation resulting in abnormal endothelial permeability and increased wall tension, resulting in progressive medial necrosis and hemorrhage.
Classification of Cystic Keratinizing Squamous Lesions of the Rat Lung: Report of a Workshop
Authors: Boorman Gary A. , Brockmann Michael , Carlton William W. , Davis John M.G. , Dungworth Donald L. , Hahn Fletcher F. , Mohr Ulrich , Reichhelm Hans-Bernhard Richter , Turusov Vladimir S. , Wagner Bernard M.
Published: April 2009

An international workshop of toxicologic pathologists reviewed cystic keratinizing squamous lesions of the rat lung. These lesions develop in response to the chronic inhalation of diverse particulate materials. Controversy exists over the biological significance of these changes and their relevance to humans. For the first time, in one place, a group of pathologists analyzed slides from all available studies. The workshop reached a consensus as to classification of these unique pulmonary tissue responses and offers diagnostic criteria for application. Although additional research is needed, this working classification scheme should serve as a practical interim approach for pathologists and regulatory agencies.
Recombinant Human Epidermal Growth Factor1-48-Induced Structural Changes in the Digestive Tract of Cynomolgus Monkeys (Macaca fascicularis)
Authors: Reindel James F. , Pilcher Gary D. , Gough Alexander W. , Haskins Jeffrey R. , De La Iglesia Felix A.
Published: April 2009

To determine the cellular effects and potential toxicity of exogenously administered recombinant human epidermal growth factor1-48(EGF1-48) in primates, intravenous bolus injections were given to 2 cynomolgus monkeys per sex at 0 (vehicle control). 10, 100, 500 (females only), and 1,000 μg/kg/day (males only) for up to 2 wk. Males given the suprapharmacologic dose of 1,000 μg/kg did not tolerate treatment and were necropsied after 5 days of dosing. All other monkeys completed the 2-wk study. Necropsy findings included enlarged, discolored, pale tan livers at 500 μg/kg and greater, firm, thickened pancreata in 500-μg/kg females, and enlarged salivary glands at all doses. Relative liver weights were increased at 500 and 1,000 μg/kg; mean salivary gland weights in all dose groups were greater than in controls. Histopathologic changes were primarily those of diffuse epithelial cell hypertrophy and hyperplasia in liver (hepatocytes and biliary tract), pancreas, salivary glands, tongue, esophagus, stomach, small and large intestine, and gallbladder. Alterations were dose-related in intensity and occurred in at least some tissues at the lowest dose. In gastric glands, colon crypts, pancreatic ducts, biliary tract, and salivary glands, differentiated epithelial cells were replaced by cells of less differentiated phenotype. These morphologic alterations were consistent with exuberant proliferation induced by this epithelial mitogen. The extent of the proliferative response in tissues of the digestive tract attests to the potency of this fragment of human EGF1-53in primates. Furthermore, the epithelial proliferation was significantly greater than that reported previously in EGF-treated rodents.
Risk Assessment of Inhaled Chloroform Based on Its Mode of Action
Authors: Wolf Douglas C. , Butterworth Byron E.
Published: April 2009

The development of scientifically sound risk assessments based on mechanistic data will enable society to better allocate scarce resources. Inadequate risk assessments may result in potentially dangerous levels of hazardous chemicals, whereas overly conservative estimates can result in unnecessary loss of products or industries and waste limited resources. Risk models are used to extrapolate from high-dose rodent studies to estimate potential effects in humans at low environmental exposures and determine a virtually safe dose (VSD). When information to the contrary is not available, the linearized multistage (LMS) model, a conservative model that assumes some risk of cancer at any dose, is traditionally employed. In the case of airborne chloroform, the dose at which an increased lifetime cancer risk of 10-6 could be calculated was chosen as the target VSD. Applying the LMS model to the mouse liver tumor data from a corn-oil gavage bioassay yields a VSD of 0.000008 ppm chloroform in the air. The weight of evidence indicates that chloroform is not directly mutagenic but, rather, acts through a nongenotoxic-cytotoxic mode of action. In this case, tumor formation results from events secondary to induced cytolethality and regenerative cell proliferation. Toxicity is not observed in rodents when chloroform is not converted to toxic metabolites at a rate sufficient to kill cells. Thus, tumors would not be anticipated at doses that do not induce cytolethality, contrary to the predictions of the LMS model. Inhalation studies in rodents show no cytolethality or regenerative cell proliferation in mouse liver at a chloroform concentration of 10 ppm as the no observed effect level (NOEL) or below. Using that NOEL and a safety factor approach, one can develop a VSD of 0.01 ppm. Integrating these data into the risk assessment process will yield risk estimates that are appropriate to the route of administration and consistent with the mode of action.
The Age-Related Difference in Bone Changes in Rats Induced by Recombinant Human Granulocyte Colony-Stimulating Factor
Authors: Suzuki Masami , Sakamaki Yoshiyuki , Miyoshi Akio , Adachi Kenji , Usami Masayoshi , Nakayama Hiroyuki , Doi Kunio
Published: April 2009

We examined the bone changes in recombinant human granulocyte colony-stimulating factor (rhG-CSF)-treated young and young adult rats in order to investigate the effect of age-related conditions of bone growth on the bone changes induced by rhG-CSF. Recombinant human G-CSF (100 and 1,000 μg/kg/day) was given to rats by daily intravenous injection for 28 days starting at the age of either 6 or 14 wk, and the hindlimb bones were evaluated histopathologically. In the young rats, bone lesions were observed in the 100- and 1,000-μg/kg groups. In the young adult rats, lesions were found only in the 1,000-μg/kg group. The lesions involved accelerated osteoclastic bone resorption and osteogenesis due to intramembranous ossification, and there was no age-related difference in these histopathological findings. However, both the incidence of bone involvement and the severity of lesions were greater in the young rats than in the dose-matched young adult rats. The results suggest that the higher dose of rhG-CSF may intrinsically induce bone lesions of a particular histopathological nature in rats regardless of their age, and the action of rhG-CSF on bone may be stronger in young growing rats than in young adults.
Poorly Differentiated Carcinoma of the Parotid Gland in a Six-Week-Old Sprague-Dawley Rat
Authors: Tsunenari Ichiro , Yamate Jyoji , Sakuma Sadashige
Published: April 2009

A poorly differentiated spontaneous tumor was found in the parotid gland region of a 6-wk-old female Sprague-Dawley rat. The tumor consisted of undifferentiated cells forming dense clusters, well-defined nests, or compact sheets. In some areas, spindle-shaped cells were arranged in a fascicular fashion. Neither ductular nor acinar structures were seen. No keratinization was observed, either. Mitotic figures were frequent. All neoplastic cells were immunoreactive for pankeratin but negative for vimentin, α-smooth muscle actin and S-100 protein. Ultrastructurally, the tumor cells were characterized by desmosome-like junctional complexes, poorly developed cytoplasmic organelles, and microvilli-like cytoplasmic processes. Based on these findings and its anatomical location, the present tumor was diagnosed as a poorly differentiated carcinoma of the parotid gland.
Intraabdominal Lymphangiosarcoma in a Fischer-344 Rat
Authors: Takahashi Kimimasa , Nakashima Nobuaki , Kuwahara Maki , Sugimoto Kayoko , Inui Kousei , Harada Takanori , Maita Keizo
Published: April 2009

A Sarcoma arising in the abdominal cavity in an aged Fischer-344 rat was studied by immunohistochemistry and electron microscopy. The white-yellow soft mass was located on the lumbosacral vertebrae, compressing adjacent parenchymal organs. The tumor was made up of spindle shaped cells situated in a background of myxoid substance and a small amount of reticulin and collagen fibers. The tumor cells grew in a loose storiform pattern and often adhered to each other by their cell processes to form ovoid or slitlike spaces. Immunohistochemically, the tumor cells were strongly positive for vimentin but negative for keratin, macrophage ED1 antigen, α-smooth muscle actin, Factor VIII-related antigen, and S100 protein. Electron microscopy demonstrated the endothelial differentiation of the tumor cells, such as occasional luminal spaces, a small number of micropinocytotic vesicles, and interdigitating junctions with desmosomes between cell processes of adjacent cells. Furthermore, its endothelial origin was suggested by the presence of electron-dense rods resembling Weibel-Palade bodies. Instead of a definitive basement lamina surrounding the tumor cells, there were extracellular thin "anchoring filaments" that were attached to the cell surface at areas of increased electron density. These findings indicate that the tumor is of lymphatic vessel type rather than blood vessel type.
Ischemic Neurons in Rat Brains After 6, 8, or 10 Minutes of Transient Hypoxic Ischemia
Authors: Radovsky Ann , Katz Laurence , Ebmeyer Uwe , Safar Peter
Published: April 2009

The incidence and distribution of ischemic (necrotic) neurons in the brains of rats 72 hr after hypoxic ischemia induced via asphyxiation is described and scored. Anesthetized Sprague-Dawley rats (10/group) were endotracheally intubated and had their airways occluded for 6, 8, or 10 min, which resulted, respectively, in approximately 3, 5, or 7 min of pulselessness (MABP < 10 mm Hg). Survival was 10/10, 9/10, and 6/10 in the 6-, 8-, and 10-min groups: deaths occurred within 1 hr after resuscitation. At 72 hr, rats were reanesthetized and their brains were perfusion-fixed with 3% buffered paraformaldehyde. Paraffin-embedded, 5-μm-thick, H&E-stained sections at 5 coronal levels of the brain had shrunken, hypereosinophilic ischemic neurons in 12 anatomic regions. Ischemic neurons were most consistently found in the lateral reticular thalamic nucleus; lateral caudoputamen; CA1 region of the hippocampus; subiculum; and, with longer asphyxia times, among cerebellar Purkinje neurons. Categorical histologic damage scores were assigned to affected regions on the basis of manual counts of ischemic neurons and summed for the whole brain. Brain histologic damage scores were significantly (p < 0.01) different for the 6-, 8-, and 10-min groups (means of 8 ± 2; 14 ± 4; and 22 ± 4). Brain regions where both the number of rats affected and ranked categorical scores for ischemic neurons increased with asphyxia time were the lateral caudoputamen and cerebellar Purkinje neurons.
Mice with Focal Pulmonary Fibrosis Caused by Monocrotaline are Insensitive to Urethane Induction of Lung Tumorigenesis
Authors: Yasuhara Kazuo , Mitsumori Kunitoshi , Shimo Takeo , Onodera Hiroshi , Takahashi Michihito , Hayashi Yuzo
Published: April 2009

To establish the characteristics of an optimized pulmonary fibrosis model, male ICR mice were given 4 weekly sc injections of 150 or 0 mg/kg monocrotaline (MCT) and maintained without further treatment for 33 wk (Experiment 1). The final mortality in the MCT group was 64%. Epithelial cells with large bizarre nuclei and an increased incidence of alveolar/bronchiolar hyperplasias were typically observed. In areas of pulmonary fibrosis, the PCNA labeling index (LI) in the alveolar/airway epithelium was significantly elevated. DNA content analysis demonstrated a larger range (4-8C) for the ploidy pattern of alveolar epithelium with large bizarre nuclei than in the normal epithelium (2C). In Experiment 2, the relationship between pulmonary fibrosis development and lung tumorigenesis was investigated. Mice were given 4 weekly sc injections of 150 and 0 mg/kg MCT, followed by a single ip injection of 1,000 or 500 mg/kg urethane (UR) on week 7, then maintained without further treatment for an additional 15 wk. UR following MCT-induced inflammatory changes, fibrosis, and epithelia with large bizarre nuclei but no tumorous lesions, in spite of the fact that treatment with UR alone caused a high incidence of pulmonary tumors. Hyperplasias were seen in all groups, but the multiplicity in the combined groups tended to be decreased by the MCT pretreatment. The present study demonstrated that this new protocol is more suitable than previous one for the experimental production of pulmonary fibrosis. Furthermore, the induction of lung tumors by UR was completely depressed in mice with MCT-induced pulmonary fibrosis, suggesting that alveolar epithelial cells are resistant to this lung carcinogen under these conditions.
Immunohistochemical Analysis of Macrophages, Myofibroblasts, and Transforming Growth Factor-β Localization during Rat Renal Interstitial Fibrosis Following Long-Term Unilateral Ureteral Obstruction
Authors: Yamate Jyoji , Okado Akiko , Kuwamura Mitsuru , Tsukamoto Yasuhiro , Ohashi Fumihito , Kiso Yasuo , Nakatsuji Shunji , Kotani Takao , Sakuma Sadashige , Lamarre Jonathan
Published: April 2009

Renal interstitial fibrosis was induced in rats by chronic unilateral ureteral obstruction (UUO). To identify the mechanisms behind the fibrosis, macrophage influx, myofibroblast involvement, and the localization of transforming growth factor-β (TGF-β, a fibrogenic cytokine) were investigated immunohistochemically in rats euthanatized at 0 (controls), 3, 6, 9, 12, and 15 days after UUO. The number of α-smooth muscle actin-positive myofibroblasts began to increase significantly in the medulla from day 3, and the development of medullary fibrosis was confirmed from day 6 by morphometric analysis. From day 9, papillary fibrosis also developed in association with an increased number of myofibroblasts. These myofibroblasts showed a parallel orientation to the mucosal surface of the pelvis. In the medulla and papilla, from day 6 the number of ED1 (primary antibody)-positive macrophages began to increase significantly. There appeared to be a relationship between macrophage influx and myofibroblast involvement. By contrast, in the cortex there was no marked increase in myofibroblasts nor development of fibrotic tissues, regardless of increased number of macrophages from day 6. Immunohistochemically, no staining for TGF-β was found in infiltrating macrophages or myofibroblasts. However, TGF-β was localized on some cortical proximal renal tubules both of normal control and obstructed kidneys in the early stages on days 3, 6, and 9, suggesting that the possible origin of TGF-β may be renal epithelia. However, the staining intensity for TGF-β on the renal epithelia tended to be weakened in advanced obstructed kidneys on days 12 and 15. The likely contribution of TGF-β to the advanced stages of UUO-induced renal fibrosis remains to be determined.
Effects of acute tryptophan depletion on prepulse inhibition of the acoustic startle (eyeblink) response and the N1/P2 auditory evoked response in man
Authors: Phillips M. A. , Oxtoby E. K. , Langley R. W. , Bradshaw C. M. , Szabadi E.
Published: April 2009

Contraction of the orbicularis oculi muscle in response to a sudden loud sound (acoustic startle response) and the N1/P2 component of the auditory evoked potential are both attenuated when a brief low-intensity stimulus is presented 30–500 ms before the ‘startle-eliciting’ stimulus (prepulse inhibition). Here, we report the effect of acute tryptophan depletion on prepulse inhibition of these responses. Thirteen males (21–52 years) participated in two sessions separated by 7 days, in which they ingested a drink containing a mixture of amino-acids, which either included (+ TP) or did not include (– TP) tryptophan, according to a balanced double-blind design. Electromyographic (EMG) responses of the orbicularis oculi muscle and N1/P2 auditory evoked potentials were recorded in a 20-min session, 6 h after ingestion of the mixture. Subjects received 40 trials in which 1-kHz sounds were presented: (i) 40 ms, 115 dB (‘pulse alone’ trials) and (ii) 40 ms, 85 dB, followed after 120 ms by 40 ms, 115 dB (‘prepulse/pulse’ trials). Mean amplitudes of the EMG response and the N1/P2 potential were derived from the pulse-alone trials and, in each case, percentage prepulse inhibition was calculated. Plasma tryptophan levels were measured from blood samples taken before and 7 h after each treatment. Under the + TP condition, both the EMG response and the N1/P2 complex showed > 60% prepulse inhibition. The – TP condition was associated with (i) significant suppression of prepulse inhibition of the EMG response, with no significant change in response amplitude and (ii) reduction of the amplitude of the N1/P2 potential, with no significant change in prepulse inhibition of this response. Tryptophan levels rose by 90 ± 15% under the + TP condition and fell by 81 ± 3% under the – TP condition. The suppression of prepulse inhibition of the acoustic startle response under the – TP condition suggests that central 5-hydroxytryptaminergic mechanisms may be involved in regulating prepulse inhibition of this response. The lack of effect of tryptophan depletion on prepulse inhibition of the N1/P2 potential suggests that different mechanisms are involved in prepulse inhibition of the startle response and the N1/P2 complex.
Depressive relapse following acute tryptophan depletion in patients with major depressive disorder
Authors: Leyton Marco , Ghadirian A. -Missagh , Young Simon N. , Palmour Roberta M. , Blier Pierre , Helmers Karin F. , Benkelfat Chawki
Published: April 2009

Acute tryptophan depletion (ATD) lowers serotonin synthesis and elicits depressive symptoms in some, though not all, remitted patients with major depressive disorder (MDD). In the present study, eight medication-free remitted patients with MDD, seasonal pattern, were tested twice, once following the ingestion of a tryptophan-containing mixture, once following ATD. ATD significantly increased Hamilton depression scores (p < 0.001). Four of the patients had a family history of psychiatric disorders: substance abuse (n= 4), mood disorders (n= 3) or Cluster B personality disorders (n= 3). The mood-lowering response to ATD was significantly greater in those patients with, than without, affected relatives (p < 0.001). These preliminary findings (1) support the hypothesis that depressed states are related to disturbed serotonin neurotransmission and (2) suggest that depressive symptoms following ATD might identify a subgroup of patients at high genetic risk for disorders associated with affective lability and dysregulated impulsecontrol, conditions thought to be related to low serotonin neurotransmission.
Evidence that long-lasting potentiation in limbic circuits mediating defensive behaviour in the right hemisphere underlies pharmacological stressor (FG-7142) induced lasting increases in anxiety-like behaviour: role of benzodiazepine receptors
Authors: Adamec Robert E.
Published: April 2009

The hypothesis that benzodiazepine receptors mediate initiation of lasting behavioural changes induced by FG-7142 was supported in this study. Behavioural changes normally induced by FG-7142 were blocked by prior administration of the competitive benzodiazepine receptor blocker, Flumazenil. When cats were subsequently given FG-7142 alone, the drug produced lasting behavioural changes in species characteristic defensive responses to rodent and cat vocal threat. FG-7142 also induced long-lasting potentiation (LLP) of evoked potentials in a number of efferent pathways from the amygdala in both hemispheres. Flumazenil given prior to FG-7142 blocked LLP in all but one of the amygdala efferent pathways, suggesting benzodiazepine receptor dependence of initiation of LLP. Three physiological changes were most closely correlated with behavioural changes. LLP in the right amygdalo-ventromedial hypothalamic (VMH) and amygdalo-periacqueductal gray (PAG) pathways coincided closely with behavioural changes, as did a reduced threshold for the right amygdalo-VMH evoked potential. Administration of Flumazenil after FG- 7142 returned defensive behaviour to pre FG-7142 baseline levels in a drug-dependent manner. At the sametime LLP only in the right amygdalo-PAG pathway was reduced by Flumazenil. LLP in other pathways and amygdalo-VMH threshold were unaltered by Flumazenil. Moreover, covariance analyses indicated that increased defensiveness depended solely on LLP in the right amygdalo-PAG. These findings support the view that maintenance of lasting increases in defensive behaviour depend upon LLP of excitatory neural transmission between amygdala and lateral column of the PAG in the right hemisphere. Moreover, FG-7142 may be a useful model of the effects of traumatic stressors on limbic system function in anxiety, especially in view of the recent data in humans implicating right hemispheric function in persisting negative affective states in post-traumatic stress disorder.
Refractory schizophrenia and atypical antipsychotics
Authors: Taylor David M. , Taylor David M. , Duncan-McConnell Denise
Published: April 2009

Treatment resistant or refractory schizophrenia is a difficult to define condition of largely unknown prevalence. For 10 years, clozapine has been the standard treatment in this condition and is recognized unequivocally as being effective. However, clozapine is sometimes poorly tolerated and has the potential for severe toxicity. Partly as a result of this, other atypicals have recently been evaluated as treatments for refractory schizophrenia. In order to evaluate the evidence base relating to the drug treatment of refractory schizophrenia, we developed a refractoriness rating based on previous work. Using this rating, we assessed all trials of atypicals in schizophrenia unresponsive to at least one drug. Overall, clozapine was consistently shown to be effective in refractory schizophrenia, even when stringently defined. Data relating to olanzapine and risperidone are equivocal at best, and there is some evidence to suggest that they are less effective than clozapine. There is essentially no cogent evidence to support the use of any other atypical in refractory schizophrenia. Clozapine remains the drug of choice in this condition.
Venlafaxine occupation at the noradrenaline reuptake site: in-vivo determination in healthy volunteers
Authors: Melichar Jan K. , Haida Abel , Rhodes Chris , Reynolds Alan H. , Nutt David J. , Malizia Andrea L.
Published: April 2009

Venlafaxine, a serotonin and noradrenaline reuptake inhibitor, is an effective antidepressant at doses of 75 mg p.o. daily and above. Preclinical and healthy volunteer studies have demonstrated that venlafaxine is more potent at the serotonin than at the noradrenaline reuptake site, with noradrenergic blocking effects being observed at doses >75 mg p.o. in man. We used the Multiple Organs Coincidences Counter and [11C] meta hydroxy ephedrine (MHED) to test whether significant occupation of cardiac sympathetic neurones was achieved in man in vivo after the acute administration of venlafaxine 75 mg p.o. in nine healthy volunteers. MHED is a tracer which binds at the noradrenaline reuptake site. This study demonstrates that the [11C]MHED signal is significantly reduced after the administration of venlafaxine 75 mg p.o. thus showing that noradrenaline reuptake blockade is observable at this dose. This effect is predominantly seen in volunteers who received > 1 mg/kg venlafaxine.