bluuebunny/crossref_metadata_2025 huggingface dataset:
Automated and Semiautomated Analysis of Rat Alkaline Phosphatase Isoenzymes
Authors: Hoffmann Walter E. , Everds Nancy , Pignatello Michael , Solter Philip F.
Published: April 2009

A semiautomated quantitative assay for rat serum alkaline phosphatase (ALP) isoenzyme determination was developed, incorporating selective precipitation of bone alkaline phosphatase (BALP) with wheat germ lectin and differential inhibition with levamisole for determination of intestinal alkaline phosphatase (IALP). The assays for each isoenzyme were linear over a broad range of activities. The within-run and between-run coefficients of variation were less than 11% for all 3 isoenzymes. Dilution of serum with saline results in an artifactual overestimation of BALP activity. Comparison of ALP and ALP isoenzyme activity in rats of various ages showed that BALP activity drops dramatically with increasing age of rats. IALP activity is greater in immature rats compared to that in mature rats. While there was no difference between male and female rats at 4 wk of age with regard to total ALP activity and activity of any of the isoenzymes, total ALP activity and activity of the individual isoenzymes were higher in males than in females at most ages over 4 wk. Gavage with corn oil resulted in increased serum IALP activity, and bile duct ligation resulted in increased liver alkaline phosphatase activity. This combined assay for the 3 ALP isoenzymes in rat serum is an efficient means of analysis of large numbers of samples and should increase markedly the specificity of serum ALP activity in identifying the target organ in toxicologic studies when serum ALP activity is increased.
Pathology and Incidence of Amelanotic Melanomas of the Skin in F-344/N Rats
Authors: Yoshitomi Katsuhiko , Elwell Michael R. , Boorman Gary A.
Published: April 2009

A total of 121 spontaneous amelanotic melanomas of the skin were identified in 70 of 11,171 male and 51 of 10,927 female Fischer-344/N rats in 63 2-yr carcinogenicity studies conducted by the National Cancer Institute's Carcinogenicity Testing Program/National Toxicology Program. Amelanotic melanomas had characteristic anatomical locations and histologic features distinguishable from Schwann cell tumors. Of the 121 tumors, 84, 19, 10, and 8 cases occurred in the pinna, eyelid, scrotum, and perianal region (anus and tail), respectively. Amelanotic melanomas originated from the dermis and consisted of spindle cells arranged in an interlacing fascicular pattern often with a perivascular orientation; epithelioid cells were rarely seen. Only the tumors arising in the pinna metastasized to the lung and/or mandibular lymph nodes. The metastatic rate was 19% (16/84) of the tumors and was clearly increased with an increase in tumor size. Most metastasizing tumors had focal areas consisting of anaplastic spindle cells with an increased number of mitosis. The tumor cells stained positive for S-100 protein but negative for melanin. Ultrastructurally, the tumors were diagnosed as amelanotic melanomas based on the identification of numerous, intracytoplasmic pre-melanosomes without melanin formation in the tumor cells which were not enveloped by pericytoplasmic basal laminae. One localized amelanotic melanoma of the pinna was successfully transplanted to the subcutaneous tissue in the flank of 3 Fischer-344/N rats.
A Simple Method for Performing Routine Histopathological Examination of the Cardiac Conduction Tissue in the Dog
Authors: Palate Bernard M. , Denoël Simone R. , Roba José L.
Published: April 2009

In standard toxicity studies, the cardiac conduction tissue is not systematically sampled and examined for histopathological changes. Most methods described use serial sectioning perpendicular to the long axis of the sinoauricular node (SAN) and atrioventricular node (AVN). Dozens of slides are needed to allow examination of a significant portion of the SAN and AVN. A simple method was developed to be used in routine histopathologic examination of the dog heart. With a plane parallel to the external wall and the upper edge of the right auricle, the SAN and its arterial supply were easily sectioned and examined. The frontal plane parallel to the interventricular crest was the most appropriate plane for observing a large portion of the AVN and the bundle of His. Based on these results, the heart of 240 dogs from toxicology studies were successfully sampled and processed utilizing this technique. An average of 5 slides per node was needed to perform a satisfactory examination of each of the SANs and AVNs.
Electron Microscopic Study on the Effect of Alternate-Day Hydrocortisone Therapy on Lymphocyte Populations in Rat Spleen and Lymph Nodes
Authors: El Fouhil Ahmed F.I. , Turkall Rita M.
Published: April 2009

The ultrastructural effects of 400 mg/M2/day of hydrocortisone sc, given alternatively from day 7 through day 19, were studied on the lymphocyte populations in the white pulp of the spleen and in the cortex of the mesenteric lymph nodes of the immunologically immature rat. Results were consistent with both a direct lytic effect of hydrocortisone on small lymphocytes of the nodular cortex of the mesenteric lymph nodes and an indirect effect on small lymphocytes of the periarterial lymphatic sheath (PALS) of the spleen supplied via the thymus. In contrast, medium-sized lymphocytes in the PALS appeared to be unaffected by hydrocortisone, while medium-sized lymphocytes of the nodular cortex of the mesenteric lymph nodes exhibited a temporary depletion of cytoplasmic organelles. Hydrocortisone appeared to depress protein synthesis in these latter cells. The decrease in numbers of lymphoblasts and plasmablasts observed in the nodular cortex of the mesenteric lymph nodes and the white pulp of the spleen is consistent with hydrocortisone interference with proliferation and differentiation of activated B cells.
The Role of Drug Metabolism in Drug Discovery: A Case Study in the Selection of an Oxytocin Receptor Antagonist for Development
Authors: L. Chiu Shuet-Ling , Thompson A. , Vincent Stella H. , Alvaro Raul F. , Huskey Su-Er W. , Stearns Ralph A. , Pettibone Douglas J.
Published: April 2009

Drug discovery is a process involving multiple disciplines and interests. During the research phase of drug discovery, usually a large number of compounds are evaluated for biological activity and toxicological potential in animal species. Various types of problems with respect to pharmacodynamics, pharmacokinetics, and toxicity are commonly encountered at this stage. Drug metabolism, as a discipline participating in a drug discovery team, can play an important role in identifying factors underlying the problems, facilitate the optimal selection of compounds for further development, provide information on metabolites for possible improvement in drug design, and contribute to the identification of the appropriate animal species for subsequent toxicity testing. During the process of evaluating oxytocin receptor antagonists for further development for treatment of preterm labor, in vivo and in vitro drug metabolism studies conducted in rats, dogs, and monkeys contributed to the selection of L-368,899 as the development candidate on the basis of pharmacokinetic and metabolism observations. The presence of active N-demethylated metabolites of two other equipotent compounds in rats and dogs was found to be the major factor responsible for the discrepancy between oral bioavailability and efficacies observed for these 2 compounds. For L-368,899, a compound that demonstrated 20-40% oral bioavailability in rats, dogs, and chimpanzees, extensive first-pass metabolism rather than absorption was determined as the major factor responsible for the poor bioavailability (< 1%) in rhesus monkeys. In vitro metabolism studies with hepatic microsomes from rats, dogs, monkeys, and humans substantiated the conclusion that the rate of hepatic metabolism of L-368,899 in monkeys is faster than in the other species.
Considerations in the Design of Toxicokinetic Programs
Authors: Cayen Mitchell N.
Published: April 2009

The objectives of toxicokinetic (TK) studies are to evaluate systemic exposure of the toxicity species to drug and/or metabolite(s) and to relate exposure to dose level and toxicological findings. The tacit assumption is that such exposure can be related to target organ toxicity. Planning a TK program involves early development (often during discovery support) of an appropriate plasma assay, assurance that the studies conform to Good Laboratory Practices, evaluating exposure at steady state, and effective collaboration among toxicologists, kineticists, and other relevant disciplines. The 3 types of TK studies-prospective, concomitant, and retrospective-each have different goals. The various stages of TK program implementation range from those during drug discovery and selection to support of chronic toxicity studies. Exposure is best expressed as the area under the plasma concentration/time curve of drug and/or metabolite(s) and, for highly protein bound drugs, is based on the unbound fraction. Although the objectives of TK programs are generally standard and the Second International Conference on Harmonization has developed TK guidelines, the programs differ among pharmaceutical companies. Some variables in program design and implementation include properties of the drug, formulation used, characteristics of the target species, ability to develop a toxicokinetictoxicodynamic relationship, strategies based on scientific/technical/philosophical considerations, dedicated resources, corporate support, and effectiveness of interdepartmental collaborations.
Concomitant Toxicokinetics: Techniques for and Interpretation of Exposure Data Obtained During the Conduct of Toxicology Studies
Authors: Dahlem Andrew M. , Allerheiligen Sandra R. , Vodicnik Mary Jo
Published: April 2009

Toxicokinetic analyses have become a routine component of preclinical toxicology studies with pharmaceutical candidates. Evaluation of plasma and/or tissue samples from animals used in toxicology studies (or concurrent satellite groups) provides information on dose proportionality, the potential for dose accumulation, and the sex and species differences in distribution and elimination. Toxicokinetic information is used by toxicologists, toxicology management, clinicians, institutional review boards, and regulatory agencies to ensure that exposure has occurred in animal species to a sufficient extent to minimize the potential risk of toxicities in humans. The requirements for descriptive toxicokinetics change depending on the stage of development of new drug candidates. Early in development, documentation of exposure in 1 species and sex of laboratory animal might be enough to justify preliminary development costs and initiation of product development. Later in development, it becomes necessary to know how new drug candidates are distributed and eliminated following subchronic and chronic administration in multiple species and both sexes. Finally, knowledge of toxicokinetics is used to help establish doses in long-term oncogenicity studies. Scientific, public, and regulatory pressures have recently dictated that the number of animals used in toxicology studies be closely monitored and minimized. Toxicokinetic evaluation of new drug candidates by a staggered sampling design is now routinely performed in our laboratories to maximize information obtained while reducing animal use.
Aerosolized Staphylococcal Enterotoxin B-Induced Pulmonary Lesions in Rhesus Monkeys (Macaca mulatta)
Authors: Mattix Marc E. , Hunt Robert E. , Wilhelmsen Catherine L. , Johnson Anthony J. , Baze Wallace B.
Published: April 2009

The pathology of aerosolized staphylococcal enterotoxin B (SEB) was studied in the nonhuman primate. Six juvenile rhesus monkeys that received multiple lethal inhaled doses of SEB developed diarrhea and vomiting within 24 hr followed by depression, dyspnea, and shock. Three of 6 animals died by 52 hr. The most striking gross lesion in all 6 monkeys was diffuse severe pulmonary edema. Histologically, edema fluid was present within the peribronchiolar, peribronchial, and perivascular interstitium, alveolar septa, and alveoli. The adventitia of pulmonary vessels was infiltrated by lymphocytes, macrophages, and fewer neutrophils. Numerous large lymphocytes with occasional mitotic figures were within pulmonary vessels, often occluding alveolar capillaries. These cells were strongly immunoreactive with monoclonal antibodies against CD3, establishing them as T cells. Ultrastructurally, endothelial cell junctions were intact, and endothelial cells and type I pneumocytes contained numerous pinocytotic vesicles. Alveolar septal interstitial spaces were expanded by edema. The mechanism of these SEB-induced pulmonary lesions was not determined. We hypothesize that cytokine production by activated T cells may have caused vascular permeability changes leading to widespread pulmonary edema and shock.
Diet, Overfeeding, and Moderate Dietary Restriction in Control Sprague-Dawley Rats: II. Effects on Age-Related Proliferative and Degenerative Lesions
Authors: Keenan Kevin P. , Soper Keith A. , Hertzog Phillip R. , Gumprecht Laura A. , Smith Peter F. , Mattson Britta A. , Ballam Gordon C. , Clark Robert L.
Published: April 2009

This study compared the effects of ad libitum (AL) overfeeding and moderate dietary restriction (DR) of 2 different diets on Sprague-Dawley (SD) rat survival and spontaneous, age-related proliferative and degenerative lesions. SD rats were fed Purina Rodent Chow 5002 or a modified Rodent Chow 5002-9 containing lower protein, fat, metabolizable energy, and increased fiber by AL or by DR at 65% of the AL amount by measurement or time (6.5 hr). At 106 wk, rats fed the 5002-9 diet AL did not have significantly improved survival over rats fed the 5002 diet AL. The 5002 diet fed DR by time (6.5 hr) improved survival for males but not females. Only DR by measurement of both diets resulted in lower mortality for both sexes. By 106 wk rats fed either diet by AL had the same brain weights as DR fed rats, but AL fed rats had greater body weight, body fat content, and increased heart, lung, kidney, liver, adrenal, thyroid, and pituitary weights that correlated with an increased incidence and severity of degenerative and/or proliferative lesions in these organs. Moderate DR delayed the progression of chronic nephropathy by delaying the early development of glomerular hypertrophy that initiates the development of glomerular sclerosis and nephron loss in AL overfed rats. Moderate DR lowered the incidence, severity, and progression of cardiomyopathy and other degenerative, age-related lesions and appeared to delay the development of reproductive senescence in SD females. The conclusion from this study is that moderate DR delayed onset and progression of degenerative lesions, and death due to cardiovascular or renal disease, and thus potentially improves the bioassay to detect compound-specific chronic toxicity.
Quantitation of Apoptotic Bodies in Rat Liver by In Situ End Labelling (ISEL): Correlation with Morphology
Authors: Wheeldon Eric B. , Williams Sally M. , Soames Anthony R. , James Neil H. , Roberts Ruth A.
Published: April 2009

Nongenotoxic hepatocarcinogens have been proposed to inhibit hepatocyte apoptosis, preventing the removal of DNA-damaged cells. Conclusive proof of this hypothesis has been hindered by the lack of a marker or stain for apoptosis suitable for high throughput counting. A method is described for the detection of apoptotic bodies (AB) in paraffin sections of rat liver using an in situ end labeling (ISEL) technique that detects DNA damage. Results of this AB quantitation were compared with routine hematoxylin-eosin (H&E)-stained sections. The number of apoptotic bodies was enhanced by the withdrawal of the rodent nongenotoxic hepatocarcinogens and hepatomitogens nafenopin (naf) or cyproterone acetate (CPA) after 7 days of daily dosing. In rat livers 24-96 hr after cessation of dosing with naf, and in control livers, an AB index of approximately 0.1% of hepatocyte nuclei was seen when stained by H&E or ISEL. However, livers examined 48 hr after cessation of 7 days of daily dosing with CPA had an AB index of approximately 1°lo hepatocyte nuclei when stained with H&E, but only approximately 0.4% hepatocyte nuclei using the ISEL technique. Thus, CPA withdrawal from the hyperplastic liver generated a wave of apoptosis in contrast to naf withdrawal where little was seen up to 96 hr after withdrawal. The differing kinetics of CPA and naf clearance may explain this discrepancy. Less apoptosis was detected by the ISEL method following CPA withdrawal; this could arise if the stage of apoptosis labeled by ISEL is shorter than the morphologically recognizable stages (using H&E). The ISEL method for the evaluation of AB indices is useful in parallel with H&E, although more validation is required before it can be used routinely for the quantitation of AB in tissue sections.
S-(1,2-dichlorovinyl)-L-cysteine-Induced Nephrotoxicity in the New Zealand White Rabbit: Characterization of Proteinuria and Examination of the Potential Role of Oxidative Injury
Authors: Davis Joe W. , Blakeman David P. , Jolly Robert A. , Packwood William H. , Kolaja Gerald J. , Petry Thomas W.
Published: April 2009

S-(1,2-dichlorovinyl)-L-cysteine (DCVC)-induced nephrotoxicity in vivo was investigated in New Zealand White rabbits. A primary emphasis in these studies was further characterization of DCVC-induced nephrotoxicity using a variety of serum and urinary analytes, including sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Additionally, the role of oxidative injury was assessed to address the dichotomy between reports indicating that such a mechanism is important in vivo and those indicating that such mechanisms do not contribute substantially to the mechanism of effects observed in vitro. Urine was collected prior to and at 8 and 24 hr after iv administration of DCVC. Serum was collected 15 min prior to and 24 hr after DCVC administration. Rabbits were euthanized 24 hr post-DCVC administration, and kidneys were fixed in formalin and further processed for light microscopic examination.

DCVC (10 mg/kg, iv) induced a 45-50-fold increase in total urinary protein excretion, a 10-15-fold increase in urinary N-acetyl-β-D-glucosaminidase concentration, plus a marked glucosuria by 24 hr postadministration. Additionally, DCVC increased serum creatinine levels by about 2-fold, with a trend toward increased blood urea nitrogen. SDS-PAGE analysis of rabbit urine confirmed the clinical finding of marked proteinuria in DCVC-treated animals, which in contrast to previously reported data was due to the presence of both low and high molecular weight proteins. Antioxidants had no significant effect on DCVC-dependent renal injury, nor was there evidence for DCVC-induced lipid peroxidation, as measured by either thiobarbituric acid-reactive substances or a commercial assay for malondialdehyde and hydroxalkenals. These latter data are consistent with previously published in vitro evidence citing no major role for lipid peroxidation in DCVC-induced nephrotoxicity.
Liver Lesions in Rats Associated with Wrapping of the Torso
Authors: Parker George A. , Gibson W. Bruce
Published: April 2009

Liver lesions were noted in control and dosed rats from a percutaneous toxicity study that involved wrapping of the torso to prevent oral ingestion following dermal application of test articles. Further investigation in a follow-up study revealed that the liver lesions were related to wrapping of the torso rather than to test-article administration because the liver lesions only appeared in wrapped animals, including sham-treated controls, but not in naive control animals. The liver lesions, which included centrilobular coagulative necrosis, inflammatory cell infiltration around biliary tracts, histiocytosis, fibrosis, and granulomatous inflammation, were compatible with infarction and associated inflammatory and reparative changes. There was no discernible pattern of involvement of specific hepatic lobes or regions of lobes. Many of the lesions were sufficiently severe to be considered life-threatening. This potentially significant complication should be considered when developing study protocols that involve wrapping of the torso of rats, and consideration should be given to inclusion of a naive control group that is not wrapped.
Immunomodulatory Effect of 2,3,7,8-Tetrachlorodibenzo-p-dioxin Tested by the Popliteal Lymph Node Assay
Authors: Fang Fan  , Pinson David M. , Rozman Karl K.
Published: April 2009

Drugs and other chemicals that have the potential to induce or exacerbate systemic autoimmune diseases in humans are of great concern. The aim of this study was to examine the immune-disregulating potential of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) by using the popliteal lymph node (PLN) assay. Chlorpromazine (CPZ) was used as a reference compound for 2 reasons: (a) CPZ is known to elicit a positive response in this assay, and (b) CPZ is a structural analogue of TCDD. Male Sprague-Dawley rats were injected subcutaneously with either TCDD or CPZ into the right hind footpad, whereas vehicle alone was injected into the contralateral footpad. Control rats were injected with vehicle in both hind footpads. Animals were sacrificed on day 7, and their PLNs were removed, weighed, and immersed in 10% formalin. The PLN weight index (the weight ratio of right PLN over left PLN) was significantly higher in both CPZ- and TCDD-treated rats than in controls. Histological examinations of PLNs in the CPZ- and TCDD-treated rats revealed similar morphological changes in both groups (e.g., mild follicular hyperplasia with no evidence of an acute inflammatory response). These results indicate that TCDD has the potential to induce or exacerbate autoimmune-like reactions. Results also suggest that drugs may be useful surrogates to study the mechanism of toxicity of environmental chemicals that cannot be administered to humans.
Effect of Methimazole on Rat Renal Carcinogenesis Induced by N-Ethyl-N-Hydroxyethylnitrosamine
Authors: Konishi Noboru , Kitamura Motokazu , Hayashi Isao , Matsuda Hirofumi , Ming Tao  , Naitoh Hiroaki , Kitahori Yoshiteru , Hiasa Yoshio
Published: April 2009

The effect of methimazole on N- ethyl-N-hydroxyethylnitrosamine (EHEN)-induced renal lesions was investigated in a medium-term initiation/promotion bioassay in male Wistar rats. EHEN-initiated rats underwent unilateral nephrectomy of the left kidney and subsequent addition of the renal tumor promoters trisodium nitrilotriacetate (NTA), potassium dibasic phosphate (PDP), or hydroquinone (HQ), alone or in combination with methimazole, to the diet for 20 wk. The addition of methimazole reduced the severity of simple and adenomatous renal hyperplasias induced by NTA, PDP, and HQ, but had no effect on the number of renal cell tumors that arose in EHEN + NTA groups. Methimazole also decreased the bromodeoxyuridine (BrdU) labeling indices, but had little effect on the expression of α2u-globulin in treated kidneys as compared to controls. It appears that methimazole exhibits antagonistic properties to nongenotoxic nephrotoxins, protecting against renal damage but not against tumorigenesis, probably arising from genotoxic insult.
Ninety-Day Feeding Study in Fischer-344 Rats of Highly Refined Petroleum-Derived Food-Grade White Oils and Waxes
Authors: Smith Jacqueline H. , Mallett Anthony K. , Priston Robert A.J. , Brantom Paul G. , Worrell Nan R. , Sexsmith Christine , Simpson Barry J.
Published: April 2009

Subchronic 90-day feeding studies were conducted in male and female Fischer-344 (F-344) rats on highly refined white mineral oils and waxes representative of those used for food applications. The goal was to help clarify the mixed results found in other toxicity studies with laboratory animals. Seven white oils and 5 waxes were fed at dietary doses of 20,000, 2,000, 200, and 20 ppm and compared with control groups on untreated diet; toxicity was assessed at 90 days and also after a reversal period of 28 days and/or 85 days. Higher molecular-sized hydrocarbons (microcrystalline waxes and the higher viscosity oils) were without biological effects. Paraffin waxes and low- to midviscosity oils produced biological effects that were inversely related to molecular weight, viscosity, and melting point; oil type and processing did not appear to be determinants. Biological effects were more pronounced in females than in males. Effects occurred mainly in the liver and mesenteric lymph nodes and included increased organ weights, microscopic inflammatory changes, and evidence for the presence of saturated mineral hydrocarbons in affected tissues. Inflammation of the cardiac mitral valve was also observed at high doses in rats treated with paraffin waxes. Further studies are required to elucidate the mechanism for the responses observed and the relevance of these inflammatory responses in the F-344 rat to other species, including humans.
Lipidosis Induced in the Dog Gallbladder by a Direct 5-Lipoxygenase Inhibitor
Authors: Molon-Noblot Sylvain , Gillet Jean-Pierre , Durand-Cavagna Geneviève , Huber Anna C. , Patrick Darryl H. , Duprat Pierre
Published: April 2009

The direct 5-lipoxygenase inhibitor L-739,010 was administered at a dose of 60 mg/kg/day per os to beagle dogs for 15 days. Histopathological examination of gallbladders from treated dogs showed epithelial vacuolation and submucosal infiltration by foamy macrophages that were positive for lipids in Sudan Black-and/or Oil Red O-stained sections. Scanning electron microscopic examination of gallbladder mucosa showed thickening of epithelial folds and multifocal epithelial membrane disruptions. Transmission electron microscopy confirmed these findings and showed mucosal epithelial cell lipid droplet accumulation and submucosal infiltration of macrophages filled with lipid droplets, myelin figures, heterophagosomes, and cholesterol clefts. These changes resembled those reportedly seen in the human gallbladder with cholesterolosis and/or chronic cholecystitis.
Chronic Administration of the Mutagenic Heterocyclic Amine 2-Amino-1-Methyl-6-Phenylimidazo[4,5-b]pyridine Induces Cardiac Damage with Characteristic Mitochondrial Changes in Fischer Rats
Authors: Takahashi Satoru , Imaida Katsumi , Shirai Tomoyuki , Wakabayashi Keiji , Nagao Minako , Sugimura Takashi , Ito Nobuyuki
Published: April 2009

Fischer-344 rats of both sexes were administered the heterocyclic amine 2-amino-1-methyl-6-phenylimidazo[4,5- b]pyridine (PhIP) for 52 wk at the dietary level of 400 ppm. Light microscopic and ultrastructural investigation of the myocardium revealed prominent changes in all PhIP-treated male and female animals investigated. These were characterized by a diffuse proliferation of markedly enlarged mitochondria, with abundant cristae and often containing lamellar bodies as inclusions. PhIP is well known to cause DNA adducts in the rat heart, and there are numerous reports of mutations in mitochondrial DNA in both humans and experimental animals being associated with very similar lesions to those observed in the present study. The results thus suggest that this heterocyclic amine induces cardiac damage by the same mechanism.
The Interactions of Diet and Colonic Microflora in Regulating Colonic Mucosal Growth
Authors: Whiteley Laurence O. , Purdon Mike P. , Ridder Greg M. , Bertram Timothy A.
Published: April 2009

The colonic mucosa can adapt its growth to alterations in diet. Metabolites from colonic microflora are frequently implicated as the primary factor in mediating the colonic mucosal response to diet; however, there is also evidence indicating that diet may have a direct effect in mediating this response. The aim of this study was to determine the role of diet, microflora, and microflora metabolites in altering the growth of the colonic mucosa. Two 28-day feeding studies were conducted using Sprague-Dawley rats. The first study compared the growth of the colonic mucosa in germ-free and conventional rats fed 6 different diets. The second study compared the growth of the colonic mucosa to the concentration of bacterial-derived short-chain fatty acids (SCFAs), bile acids, and ammonia. The diets that were fed consisted of (1) AIN-76a diet without dietary fiber; (2) standard AIN-76a diet, which contained 5% cellulose; (3) AIN-76a diet with 5% guar gum; (4) a "Western" human diet with 20% fat and 10% cellulose; (5) AIN-76a diet formulated to mimic Diet 4 in fat content but with 2.5% cellulose; and (6) Purina Rodent Chow. Quantitative volumetric and stereologic analysis was used to assess changes in total colonic mucosal volume as a measure of mucosal growth. In germ-free rats, Diets 2-4 and 6 induced a significant increase (18-38%) in mucosal volume compared to Diet 1. In conventional animals, only Diets 4 and 6 induced a significant increase (up to 63%) in mucosal volume compared to Diet 1. Relative to the germ-free animals, only conventional animals on Diets 4 and 6 had an increase in mucosal volume. The increases in mucosal volume in Diets 4 and 6 were not consistently associated with increased SCFAs, ammonia, or bile acids. There was a wide range in the colonic concentrations of SCFAs (2-fold), ammonia (6-fold), and bile acids (10-fold). The presence of colonic microflora in and of itself does not lead to enhanced colonic mucosal growth. Rather, there are unique interactions between specific types of diet and microflora that lead to a growth-promoting effect. This effect could not be explained by alterations in the concentration of SCFAs, ammonia, or bile acids in colonic contents.
N-Methyl-N'-Nitro-N-Nitrosoguanidine-Induced Neoplasms in Medaka (Oryzias latipes)*1
Authors: Bunton T.E. , Wolfe Marilyn J.
Published: April 2009

To test the sensitivity of the small fish species Oryzias latipes to the direct-acting carcinogen N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), medaka were exposed at 15 days of age to 30 mg/L for 1 hr and followed for up to 16 mo. One hundred neoplasms were diagnosed in 84 of 213 exposed fish, with approximately equal percentages in males and females. Many neoplasms (62%) were of mesenchymal origin and were categorized as blood vascular neoplasms (hemangioma and hemangiosarcoma), invasive sarcomas, and scale-associated neoplasms. Invasive sarcomas included rhabdomyosarcoma, fascial sarcoma, hemangiopericytoma, and undifferentiated sarcoma. A scale-associated neoplasm, termed lepidocytoma, was an unusual neoplasm of scale anlage. Thyroid follicular neoplasms, with a 100% incidence in males, and pancreatic acinar carcinoma were the most common epithelial tumors. Neoplasms of the gills, swim bladder, and olfactory epithelium were also seen as well as teratoma with mixed epithelial and mesenchymal components. The study showed a broad range of neoplasms induced by MNNG in medaka, with a tissue distribution that might support direct action on exposed tissues.
Brief Communication
Authors: Crist Keith A. , Fuller Raymond D. , Chaudhuri Bina , Chaudhuri Prabir , Ming You
Published: April 2009

P53 aberrant protein expression and mutation is a component of many human tumors but less information is available regarding involvement in relevant animal models. We have examined invasive mammary epithelial neoplasms for p53 aberrant protein expression from Sprague-Dawley rats induced by two intrajugular injections of N-methyl- N-nitrosourea (NMU, 50 mg/kg), 7 days apart, beginning at 44-49 days of age. Positive nuclear staining was observed in 8/10 mammary tumor frozen sections using PAb 421, a mouse monoclonal antibody, compared to a mixed mouse IgG negative control antibody. Paraffin sections from two additional sets of similarly induced rat mammary tumors also showed positive nuclear staining (22/37 tumors) with CM-5, a rabbit polyclonal antibody. Our results indicate that elevated cellular content of p53 is a common event in invasive palpable mammary tumors induced by NMU in this dual-injection model system.
Cadmium-Induced Dental Lesions in Ovariectomized Rats
Authors: Katsuta Osamu , Hiratsuka Hideaki , Matsumoto Junko , Tsuchitani Minoru , Umemura Takashi
Published: April 2009

The effects of cadmium chloride on both incisor and molar teeth of ovariectomized female rats were studied histopathologically. The rats were injected intravenously with the compound at doses of 1.0 and 2.0 mg/kg, 5 days/wk. Six rats per group were sacrificed at 4, 8, and 13 wk.

Discoloration of the incisors was observed in the rats of the 2.0-mg/kg group from 8 wk. Histopathologic examination of the incisor demonstrated decreased iron-containing pigment in ameloblasts and destruction of the enamel organ. These changes were accompanied with accumulation of cadmium and loss of iron in the teeth. Necrosis of the dental pulp occurred from the coronal end of both the incisor and molar teeth extended to the apical, deep portion of the teeth. The dental pulp of the molar teeth, which is shorter than that of the incisor, was mildly affected by cadmium intoxication. These findings suggested that intradental ischemia due to cadmium toxicity may have contributed to the development of the pulpal necrosis.
The Synovial Membrane, Liver, and Tongue: Target Organs for a Ricin A-Chain Immunotoxin (ZD0490)
Authors: Westwood F. Russell , Jones David V. , Aldridge Andrew
Published: April 2009

ZD0490 is an immunotoxin consisting of a mouse monoclonal antibody conjugated to recombinant ricin A-chain (rRAC). It was developed at Zeneca Pharmaceuticals as a treatment for certain antigen-bearing tumors. During safety evaluation studies in rats, a number of reversible inflammatory changes were seen. The synovial membranes of articular joints showed a marked degeneration and necrosis with an associated inflammation. When of mild severity only the synovial membrane was involved, but when more severe many adjacent tissues including the surface of the articular cartilage were affected. Some nonspecific skeletal muscle toxicity occurred. However, tongues from the intravenously (tail) dosed rats consistently showed inflammation specifically located in the ventral subepithelial area with myocyte degeneration and necrosis. Also, hepatic peliosis primarily located in the subcapsular areas was induced. Studies with rRAC alone indicated that ricin A-chain (RAC) is the component responsible for these findings. It is suggested that cells of a macrophage type with the ability to specifically bind RAC may at least in part determine the location and nature of the changes seen.
A Review of the Hepatic Tumors Related to Mixed-Function Oxidase Induction in the Mouse
Authors: Butler W.H.
Published: April 2009

Mixed-function oxidase (MFO) induction in the mouse liver results in a rapid and sustained centrilobular hypertrophy associated with a hyperplastic response. In many studies, the long-term sequela of prolonged exposure is an increased incidence of lesions considered to be adenomas. Studies have shown in aged control mice that the burden of adenomas usually consists of lesions with basophilic cytoplasic staining and a uniform population of hepatocyte nuclei. With long-term feeding of MFO inducers, there is an additional burden of lesions diagnosed as adenomas having a different histological appearance with increased eosinophilic cytoplasm and pleomorphic nuclei. The incidence of hepatocarcinomas usually is not modified by the increased incidence of eosinophilic adenomas. Studies into the behavior of the eosinophilic lesions show that the hepatocytes approximate in their behavior to normal and not neoplastic cells. It is suggested that these lesions should not be considered a carcinogenic response to the chemical.
Equivocal Evidence of Complete Carcinogenicity after Repeated Infection of Syrian Hamsters with Opisthorchis viverrini
Authors: Thamavit Witaya , Tiwawech Danai , Moore Malcolm A. , Ito Nobuyuki , Shirai Tomoyuki
Published: April 2009

The effects of repeated infection with Opisthorchis viverrini on liver lesion development in male and female Syrian hamsters were investigated over a 1-yr period. Ten monthly intragastric applications of 50, 25, 13, or 0 parasite metacercariae resulted in pronounced proliferative and inflammatory lesions involving the first- and second-order ducts in response to the presence of adult worms. Despite the development of small numbers of putative preneoplastic areas of cholangiofibrosis and morphologically altered hepatocellular foci, no neoplastic lesions were evident at sacrifice after 1 yr. The results thus suggest that parasite infestation is itself not strongly carcinogenic if at all but, rather, that it exerts a marked promoting influence on cholangiocellular and hepatocellular tumor development in the hamster via chronic irritation and increased cell turnover.
Third International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use— A Toxicologist's Perspective
Authors: Smith Peter E
Published: April 2009

This overview provides a perspective on the ICH process and outcomes based on the third international conference held in Yokohama, Japan, in November/December 1995. It is an attempt to provide awareness of this process to the toxicologic pathology community from the point of view of a practicing toxicologist and meeting attendee and to consolidate much of the information on this process in one place. The ICH process is an active, and ongoing process to improve the standardization of safety assessment testing throughout the world, and further changes will no doubt have occurred by the time this document is published.
Pathology of Perbutylated-N-Butyl-1-Deoxynojiromycin (An α-Glucosidase-1 Inhibitor) in Sprague-Dawley Rats
Authors: Khan Kanswar Nasir M. , Snook Sandra S. , Semler David E. , Baron David A. , Alden Carl L.
Published: April 2009

Perbutylated- N-butyl-1-deoxynojiromycin (p- N-butyl-DNJ, SC-49483), an α-glucosidase-1 inhibitor, is a candidate anti-HIV agent targeted against viral glycoprotein processing in host cell endoplasmic reticulum. The potential toxicity of this compound was evaluated in Sprague-Dawley rats after 4, 13, or 26 wk of oral administration at doses ranging from 300 to 3,670 mg/kg/day. In these studies, the target organs of p- N-butyl-DNJ effects were thyroid gland, salivary gland, stomach, and pancreas. The most prominent histologic change in these organs was the presence of clear or lightly eosinophilic vacuoles in the cytoplasm of thyroid follicular cells, gastric chief cells, salivary gland acinar cells, and exocrine pancreatic acinar cells. Ultrastructurally, these vacuoles were consistent with dilated rough endoplasmic reticulum, which sometimes contained homogeneously stained, moderately electron-dense material. The vacuoles in thyroid follicular cells contained pale eosinophilic colloidlike material consistent with accumulated thyroglobulin, as shown by immunohistochemical staining methods. The biological functions of these organs were not adversely affected as evidenced by the absence of clinical signs and the results of selected hormonal analyses. The morphologic changes were completely reversed after a 4-wk recovery period. The incidence and severity of histologic changes were decreased after 13 and 26 wk of treatment compared to 4 wk of treatment, indicating an attenuation of the host response or adaptation to the prolonged p-N-butyl-DNJ administration. We believe that morphologic changes in thyroid follicular cells, salivary gland acinar cells, pancreatic acinar cells, and gastric chief cells were the result of nonspecific inhibition of host α-glucosidase(s) by p- N-butyl-DNJ, causing clinically silent perturbation in host cell glycoprotein processing and/or glycoprotein transport.
Case Report
Authors: Logan Ava C. , Khan Kanwar Nasir M.
Published: April 2009

Two sexually mature marmosets (Callithrix jacchus) showing clinical signs similar to those seen in wasting marmoset syndrome (weight loss, decreased muscle mass, and alopecia) were evaluated for clinical and anatomic pathologic changes. The most prominent clinical pathologic alterations included macrocytic normochromic anemia, hypoproteinemia, hypoalbuminemia, elevated serum aspartate aminotransferase and alkaline phosphatase levels, and previously unreported changes of thrombocytosis. The principal gross and histopathologic finding was chronic colitis, which appeared to be the most important contributing factor to the development of wasting syndrome in these marmosets.
Possible Mechanisms Underlying the Antiaging Actions of Caloric Restriction
Authors: Masoro Edward J.
Published: April 2009

Restricting the food intake of mice and rats to well below that of ad libitum-fed animals markedly slows the aging processes. This action is reflected in an increase in longevity, a decrease in the age-associated increase in age-specific mortality rate, the maintenance of the physiological processes in a youthful state even at advanced ages, and the delaying of the onset or slowing of the progression or both of most age-associated diseases. The dietary factor responsible is the reduction in energy (caloric) intake. Many hypotheses have been proposed regarding mechanisms underlying this antiaging action. Hypotheses relating the antiaging action to the retardation of growth and development, the reduction of adipose mass, and the reduction of metabolic rate have been found to be wanting. Two of the proposed hypotheses have some evidence in their support. One involves the altered metabolic characteristics of glucose fuel use and of oxidative metabolism. The other relates to the enhanced ability of the rodents restricted in food intake to cope with challenges, which in turn has been linked to the glucocorticoid system and to the heat-shock protein system.
Neuronal and Muscular Inclusions in Rats with Hindlimb Dysfunction after Treating with Difluorobenzhydrylpiperadine
Authors: Yamanaka Yoshihiro , Shimada Tetsu , Mochizuki Rika , Suzuki Yoko , Takenouchi Kazuya , Takeda Toshiaki , Uno Hiroshi , Izawa Yoshihiro , Fujiwara Kosaku
Published: April 2009

Rats showing an ataxic gait induced by 20 wk of treatment with 0, 30, or 60 mg/kg of difluorobenzhydrylpiperadine (DFBP), a detriazinyl metabolite of almitrine, were examined by light microscopy and transmission electron microscopy. Vacuolar degeneration associated with lamellar inclusions was observed in musculus soleus and m. interossei of the hindlimbs in DFBP-treated rats. The inclusions were also produced within sensory neurons, satellite and Schwann cells, and vascular endothelial cells of thoracic and lumbar dorsal root ganglia as well as muscle spindles of affected muscles. Membrane-bound vacuoles containing electron-dense granules were seen in the peripheral nerves. This study demonstrated neuronal and muscular toxicity of DFBP in rats.
Diagnostic Criteria for Degenerative, Inflammatory, Proliferative Nonneoplastic and Neoplastic Liver Lesions in Medaka (Oryzias latipes): Consensus of a National Toxicology Program Pathology Working Group
Authors: Boorman Gary A. , Botts Suzanne , Bunton Tracie E. , Fournie John W. , Harshbarger John C. , Hawkins William E. , Hinton David E. , Jokinen Michael P. , Okihiro Mark S. , Wolfe Marilyn J.
Published: April 2009

Diagnostic criteria are presented for degenerative, inflammatory, nonneoplastic proliferative, and neoplastic lesions in the liver of medaka (Oryzias latipes), a small fish species frequently used in carcinogenesis studies. The criteria are the consensus of a Pathology Working Group (PWG) convened by the National Toxicology Program. The material examined by the PWG was from medaka exposed to N-nitrosodiethylamine for 28 days, removed to clean water, and sacrificed 4, 6, or 9 mo after initiation of exposure. Degenerative lesions included hepatocellular intracytoplasmic vacuolation, hepatocellular necrosis, spongiosis hepatis, hepatic cysts, and hepatocellular hyalinization. Inflammatory lesions consisted of granulomas, chronic inflammation, macrophage aggregates, and focal lymphocytic infiltration. Nonneoplastic proliferative lesions comprised foci of cellular alteration (basophilic focus, eosinophilic focus, vacuolated focus, and clear cell focus) and bile duct hyperplasia. Neoplastic lesions included hepatocellular adenoma, hepatocellular carcinoma, cholangioma, and cholangiocarcinoma. Two lesions composed mainly of spindle cells were noted, hemangiopericytoma and spindle cell proliferation. Rather than being an exhaustive treatment of medaka liver lesions, this report draws from the published literature on carcinogen-induced liver lesions in medaka and other fish species and attempts to consolidate lesion criteria into a simplified scheme that might be useful to pathologists and other researchers using medaka lesions for risk assessment or regulatory purposes.
The Effect of Erythritol on the Stability of γ-glutamyl Transpeptidase and N-acetyl Glucosaminidase in Human Urine
Authors: Loeb Walter F. , Das Saroj R. , Trout J. Richard
Published: April 2009

In toxicology studies and clinical trials of erythritol, treated animals and human subjects had higher urine γ-glutamyl transpeptidase [γ-glutamyl transferase (γ-GT)] than untreated controls. It has previously been reported that γ-GT activity in frozen urine decreases with time; therefore, a study was undertaken to examine the effects of storage temperature, time, and the presence of erythritol on the stability of γ-GT and N-acetyl glucosaminidase in human urine. In this study, it was found that the rate of decrease of the activity of γ-GT is much greater at -20°C than at -70°C. Variation in the storage temperature of the frozen urine is particularly deleterious to γ-GT. The addition of erythritol in a concentration of 5% reduces this decrease. Approximately 15% of N-acetyl glucosaminidase activity is lost in the initial process of freezing the urine. Thereafter, conditions of temperature, time, and the presence or absence of erythritol account for little additional loss of activity.
Spontaneous Iron Overload in Sprague-Dawley Rats
Authors: Masson Régis , Roome Nigel Oliver
Published: April 2009

In a review of the toxicological studies performed in our laboratory during the period 1986-1995, we occasionally observed significant iron overloading in the liver. Liver tissue was examined by light and electron microscopy, and the results were analyzed by sex and age (7, 9, 11, 19, 31, 59, and 111 wk). The intensity of iron overload increased with age: the accumulation began in pericanalicular siderosomes of periportal hepatocytes and extended progressively to the entire lobule and also to nonhepatocytic cells (Kupffer cells in sinusoids and macrophages around bile ducts in portal tracts) and occasionally with distortion of sinusoids by sideroblastic nodules and moderate enlargment of portal tracts in the oldest animals. No significant inflammatory infiltrates, degeneration, necrosis or fibrosis were noted. Hepatocyte pigmentation alone was prominent at 9 and 11 wk. The frequency of pigmentation of parenchymal and nonhepatocytic cells increased from 9 wk for females; in males, this was seen only at 111 wk. The intensity of pigmentation of nonhepatocytic cells versus parenchymal cells increased with aging. The frequency of those different types of iron overloading was higher for females up to 111 wk. The pathology of spontaneous iron overloading in the Sprague-Dawley rat, described here in spite of differences, has some similarities to that of human hereditary hemochromatosis.