bluuebunny/crossref_metadata_2025 huggingface dataset:
The Measurement of Renal Injury
Authors: Loeb Walter F.
Published: September 2009

The classical assessment of nephrotoxicity by the measurement of serum urea nitrogen and creatine is insensitive and nonspecific. Selection from a battery of clinical pathology tests allows the sensitive and specific measurement of renal dysfunction and injury. This requires the collection of urine specimens of good technical quality, with appropriate preservation. Interaction of the test article with urinary enzymes should be evaluated prior to their measurement for the assessment of tubular injury. This enables the localization and quantification of the injury within the regions of the nephron. High resolution proton nuclear magnetic resonance spectroscopy, a technique for measurement of low molecular weight metabolites, shows great promise for the evaluation of renal tubular injury in toxicologic studies.
In VitroMethods of Assessing Renal Damage
Authors: Lash Lawrence H.
Published: September 2009

Freshly isolated and primary cultures of rat kidney cells derived from specific nephron segments can be useful in vitro models for studying processes such as drug metabolism, membrane transport, and biochemical mechanisms of chemically induced toxicity. Proximal tubular (PT) and distal tubular (DT) cells were isolated from rat renal cortex by collagenase perfusion and Percoll density-gradient centrifugation. Oxidants produced glutathione (GSH) oxidation and lipid peroxidation and were markedly more cytotoxic to DT cells than to PT cells. Similarly, alkylating agents that target soft nucleophiles such as GSH and protein sulfhydryls were more toxic to DT cells than to PT cells, whereas an alkylating agent that targets hard nucleophiles was equally cytotoxic in the 2 cell types. DT cells were also more sensitive to brief periods of oxygen deprivation and were markedly more susceptible to ATP depletion by treatment with iodoacetate and cyanide than were PT cells. Serum-free, hormonally defined conditions have been optimized for primary culture of rat renal PT and DT cells to maintain differentiated function for up to 9 days. Primary cultures exhibited similar susceptibilities as freshly isolated cells to acute injury from chemical toxicants and the cultures express several isoforms of cytochrome P-450. These studies show that freshly isolated and primary cultures of rat renal PT and DT cells can be used to study both short-term and long-term responses to toxic chemicals.
In Situ Hybridization in the Study of Remodeling in Proliferative Glomerulonephritis
Authors: Barnes Jeffrey L.
Published: September 2009

In situ hybridization combined with immunohistochemistry provides a powerful tool to study the temporal and spatial relationships between cellular sources of mRNA and localization of translated protein in normal biologic and pathologic processes. In this symposium, techniques in probe selection for the detection of mRNA in normal kidney and renal disease were discussed. Examples of the application of in situ hybridization in the study of renal disease were demonstrated using a model of proliferative glomerulonephritis induced by habu snake venom. This model follows an accelerated course of remodeling involving mesangial cell migration, proliferation, and extracellular matrix synthesis. The cellular sources and temporal expression of 2 adhesive proteins, fibronectin and thrombospondin, known to have a role in cell remodeling during embryogcnesis and wound healing, were examined and compared to mesangial cell behaviors during the course of habu venom-induced glomerulonephritis. Mesangial cell migration in early lesions was associated with thrombospondin and fibronectin derived from platelets or macrophages. Thrombospondin mRNA and protein peaked at 48 hr after habu venom and were associated with mesangial cell proliferation; but thrombospondin mRNA and protein declined at 72 hr when expression of collagen type IV and laminin mRNA and protein peaked. Mesangial cell expression of fibronectin first appeared at 48 hr, and peaked at 72 hr after habu venom. Thus, mesangial cell migration was associated with exogenous fibronectin and thrombospondin derived from platelets or macrophages. Mesangial cell expression of thrombospondin was associated with migration and proliferation, whereas, expression of fibronectin was associated with proliferation and matrix synthesis. These results suggest distinctive temporal and spatial roles for thrombospondin and fibronectin in remodeling during glomerulonephritis and illustrate the utility of in situ hybridization and immunohistochemistry in the detection of cellular sources of translated proteins.
Examination of the Mechanisms of Action of Diverse Cytoprotectants in Renal Cell Death
Authors: Waters Shayla L. , Schnellmann Rick G.
Published: September 2009

Glycine, strychnine, muscimol, allopregnanolone, and pregnenolone sulfate act in the late phase of renal cell injury, block Cl−influx and cell lysis induced by the mitochondrial inhibitor antimycin A, and promote the recovery of respiration and ion transport following hypoxia/reoxygenation. However, the mechanism of action of these compounds has not been completely elucidated. Recently, we have shown that calpains are critical mediators of renal cell death produced by diverse toxicants and that antimycin A exposure results in calpain translocation from the cytosol to the membrane fraction that is temporally associated with Cl−influx and precedes cell death/lysis. The current study examined the effects of a group of diverse cytoprotectants on calpain activity and determined if calpain inhibition plays a role in the cytoprotection produced by these compounds. The cytoprotection produced by glycine, strychnine, muscimol, allopregnanolone, and pregnenolone sulfate in rabbit renal proximal tubules exposed to antimycin A was associated with the inhibition of antimycin A-induced calpain translocation. None of the cytoprotectants had a direct effect on calpain activity. All of the cytoprotectants decreased calcium-ionophore-induced cell death. Glycine, strychnine, and muscimol also blocked antimycin A mediated extracellular Ca2+influx. These data suggest that the cytoprotective mechanism of action of glycine, strychnine, and muscimol involves the inhibition of antimycin A mediated extracellular Ca2+influx as well as calpain translocation and associated Cl−influx. In contrast, the mechanism of action of the neurosteroids results only from the blockade of calpain translocation and associated Cl−influx.
Renal Papillary Necrosis—40 Years On
Authors: Bach Peter H. , Thanh Nguyen Thi Kim
Published: September 2009

Analgesics and nonsteroidal anti-inflammatory drugs (NSAIDs) are well recognized as a major class of therapeutic agent that causes renal papillary necrosis (RPN). Over the last decade a broad spectrum of other therapeutic agents and many chemicals have also been reported that have the potential to cause this lesion in animals and man. There is consensus that RPN is the primary lesion that can progress to cortical degeneration; and it is only at this stage that the lesion is easily diagnosed. In the absence of sensitive and selective noninvasive biomarkers of RPN there is still no clear indication of which compound, under what circumstances, has the greatest potential to cause this lesion in man. Attempts to mimic RPN in rodents using analgesics and NSAIDs have not provided robust models of the lesion. Thus, much of the research has concentrated on those compounds that cause an acute or subacute RPN as the basis by which to study the pathogenesis of the lesion. Based on the mechanistic understanding gleaned from these model compounds it has been possible to transpose an understanding of the underlying processes to the analgesics and NSAIDs. The mechanism of RPN is still controversial. There are data that support microvascular changes and local ischemic injury as the underlying cause. Alternatively, several model papillotoxins, some analgesics, and NSAIDs target selectively for the medullary interstitial cells, which is the earliest reported aberration, after which there are a series of degenerative processes affecting other renal cell types. Many papillotoxins have the potential to undergo prostaglandin hydroperoxidase-mediated metabolic activation, specifically in the renal medullary interstitial cells. These reactive intermediates, in the presence of large quantities of polyunsaturated lipid droplets, result in localized and selective injury of the medullary interstitial cells. These highly differentiated cells do not repair, and it is generally accepted that continuing insult to these cells will result in their progressive erosion. The loss of these cells is thought to be central to the degenerative cascade that affects the cortex. There is still a need to understand better the primary mechanism and the secondary consequences of RPN so that the risk of chemical agents in use and novel molecules can be fully assessed.
Mechanisms of Chemically Induced Renal Carcinogenesis in the Laboratory Rodent
Authors: Hard Gordon C.
Published: September 2009

Laboratory studies with classical renal carcinogens in the rat and mouse, as well as research investigation with some of the chemicals proving positive for the kidney in National Toxicology Program carcinogenicity bioassays, have demonstrated the existence of a range of diverse mechanisms underlying rodent kidney carcinogenesis. The classical carcinogens used as experimental models for studying renal tumor pathogenesis, such as the nitrosamines, are genotoxic and interact directly with DNA, forming DNA adducts with mutagenic potential. In contrast, potassium bromate and ferric nitrilotriacetate (Fe-NTA), also effective renal carcinogens, appear to cause indirect damage to DNA mediated by oxidative stress. A number of nongenotoxic chemicals are associated with epigenetic renal tumor induction in rodents, and the activity of these tends to involve prolonged stimulation of cell proliferation throughout the duration of exposure. This mode of action reflects a sustained regenerative response, either due to direct chemical toxicity to the tubule cells, as with chloroform, or to indirect cytotoxicity associated with lysosomal overload, as in α2u-globulin accumulation in male rats resulting from the administration of such chemicals as d-limonene and tetrachloroethylene. The histopathologic nature of hydroquinone renal carcinogenesis suggests that an additional epigenetic pathway to renal tubule tumor formation in rats may be through chemical-mediated exacerbation of, and interaction with, the age-related spontaneous renal disease, chronic progressive nephropathy. These various mechanistic pathways have implications for the nature of the induced cancer process with respect to tumor incidence, latency, malignancy, and sex predisposition.
Molecular Genetics of Renal Carcinogenesis
Authors: Walker Cheryl
Published: September 2009

Renal cell carcinoma (RCC) is the most common tumor of the adult kidney, accounting for 85% of renal neoplasms. RCC is heterogeneous in appearance, displaying diverse histologic and cytologic characteristics, with the clear cell variant being the most common. Individuals at high risk for this disease include persons with end-stage renal disease, those with hereditary predispositions such as von Hippel-Lindau syndrome (VHL) or tuberous sclerosis (TSC), and individuals with significant environment exposures such as smoking or analgesic abuse. Recently, several of the genetic targets for alterations involved in the development of human RCC have been identified. Solid RCC of the clear cell type is associated with alterations in the VHL tumor suppressor gene and hereditary papillary RCC is associated with alterations of the c-met protooncogene. In the rat, the most commonly seen tumors are of the non-clear cell type and it is the Tsc-2 tumor suppressor gene, rather than the VHL tumor suppressor gene, that appears to be the primary target for both spontaneous and carcinogen-induced mutations in these animals. These data suggest that different variants of RCC have distinct molecular etiologies and that there are species-specific determinants that modulate the involvement of specific tumor suppressor genes in RCC. Interestingly, many of the genes involved in RCC also play significant roles in kidney development. The Wilm's tumor suppressor gene, WT-1, and Pax-2 regulate the mesenchymal epithelial transition that occurs during nephrogenesis and both these genes exhibit altered expression patterns and/or are mutated in renal tumors. Other genes such as c-met and its ligand hepatocyte growth factor are also involved in normal development and tumorigenesis, suggesting that tumors arise as a result of altered functions that are reflective of events that occur during nephrogenesis.
Effect of Papillotoxic Agents on Expression of Cyclooxygenase Isoforms in the Rat Kidney
Authors: Khan Kanwar Nasir M. , Alden Carl L. , Gleissner Stephanie E. , Gessford Mary K. , Maziasz Timothy J.
Published: September 2009

Inhibition of renal vasodilatory prostaglandins (PGs) and secondary ischemia due to inhibition of cyclooxygenase (COX) activity has been suggested as a possible mechanism for development of analgesic-related renal papillary necrosis (RPN) in rats. Recently, it has been shown that COX exists in two related but unique isoforms, COX-1 and COX-2. It is unclear what potential roles these isoforms play in the maintenance of blood flow in the renal papilla or genesis of RPN. We evaluated the effect of 2 papillotoxic agents, including a nonsteroidal anti-inflammatory drug, indomethacin, and a chemical agent, 2-bromoethanamine hydrobromide (2-BEA), on COX-1 and COX-2 in the renal papilla as a means of assessing what changes occur in the expression of these isoforms during the development of RPN. Female Wistar rats ≃10–17 wk old were treated with either indomethacin (75 mg/kg, single dose, or 10 mg/kg/day for 5 days) or 2-BEA (100 mg/kg/day for 4 days) to create lesions of RPN. In this study, a single 75-mg/kg dose of indomethacin did not cause light microscopic changes of RPN. However, RPN was observed in animals administered indomethacin at 10 mg/kg/day for 1 wk or 2-BEA for 5 days. The immunohistochemical analyses of kidneys showed that both COX-1 and COX-2 were present in the renal papilla of control rats. In animals treated with indomethacin (75 mg/kg), a slight to moderate decrease in both isoforms was observed in essentially normal renal papillary cells within 2 hr, that was followed by an increase in COX-2 immunoreactivity in the renal papilla, macula densa, and thick ascending limbs (both 10-and 75-mg/kg animals). This COX-2 immunoreactivity was greatest in animals with concomitant indomethacin-induced gastrointestinal injury, suggesting a possible role of inflammatory cytokines in COX-2 induction. No changes in the expression of COX isoforms in the intact papilla occurred as a result of 2-BEA; however, cells undergoing degeneration and necrosis lost immunoreactivity to both COX isoforms. The possible mechanism that leads to an initial decrease in COX immunoreactivity in indomethacin-treated animals is not known; however, a reversible ultrastructural change in the papillary cells cannot be ruled out. This decrease in COX isoforms in the renal papilla may contribute to the development of RPN through the loss of vasodilatory PGs.
Serotonin (5-HT1A-Receptor) Agonist-Induced Collecting Duct Vacuolation and Renal Papillary Necrosis in the Rat
Authors: Rinke Matthias , Bomhard Ernst M. , Hildebrand Heinz , Leser Karl H. , Loof Ingo , Ruehl-Fehlert Christine
Published: September 2009

General anxiety in humans is treated with azaspirodecanedions, which act through a reduction of serotonin transmission. Ipsapirone also represents a serotonin (5-HT1A) receptor agonist and was under development as an anxiolytic drug. Histopathologic evaluation of animal experiments revealed cellular swelling and/or vacuolation of renal papillary and medullary collecting duct (MCD) epithelium in rats but not in dogs or mice. The changes ensued already after 1 wk of dosing and were first localized in the inner MCDs. Longer treatment periods showed that these changes proceeded from proximal to distal, approaching the papillary collecting ducts. The changes were most likely the result of altered hemodynamics in the papillary tip. Swelling resulted in partial or total papillary necrosis in some cases. Furthermore, rats treated with ipsapirone showed a sharp and transient rise in urinary endothelin excretion. Concomitantly, urinary PGE2levels were elevated. In contrast, no elevated levels of endothelin were detected in urine samples of patients from a volunteer study, leading to the conclusion that the human kidney is not susceptible to the ipsapirone-induced alterations seen in the collecting ducts of rats.
Effect of Chronic Growth Hormone Administration on Skeletal Muscle in Dogs
Authors: Molon-Noblot Sylvain , Laroque Philippe , Prahalada Srinivasa , Stabinski Lea G. , Hoe Chao-Min , Peter Chennekatu P. , Duprat Pierre , Van Zwieten Matthew J.
Published: September 2009

Administration of growth hormone (GH) results in increased body weight gain in dogs. Increased body weight gain is believed to be a result of the trophic effect of GH on the musculoskeletal system. However, edema is one of the side effects described in man following exogenous GH administration. Thus, the objective of this study was to determine if the expected increased weight gain in GH-treated dogs is a result of increased muscle mass. Porcine growth hormone (pGH), administered subcutaneously to beagle dogs at doses of 0.025, 0.1, and 1 IU/kg/day for 14 wk, resulted in elevated serum GH and insulin-like growth factor-1 (IGF-1) levels (see accompanying paper, Prahalada et al). This was associated with a significant increase in body weight gain and weights of the cranial tibialis muscle in both male and female dogs. The increased muscle mass likely contributed to the significant increase in body weight gain seen in both sexes. Quantitative analysis of skeletal muscle sections stained for ATPase activity showed increases in type I (slow twitch) and type II (fast twitch) myofiber sizes in mid- and high-dose males and in high-dose females. The ratio of type I and type II muscle fibers remained unchanged. Hypertrophic myofibers were enlarged but had a normal histologic and ultrastructural organization when observed by light and transmission electron microscopy. The results of this study have demonstrated that increased muscle mass in pGH-treated dogs is related to hypertrophy of muscle fibers and not due to edema. Exogenous GH administration has an anabolic effect on skeletal muscle in dogs.
Ocular Irritation: Pathological Changes Occurring in the Rat with Surfactants of Unknown Irritancy
Authors: Maurer James K. , Parker Ronald D. , Carr Gregory J.
Published: September 2009

We believe the development and validation of in vitro alternatives to eliminate the need to use animals in ocular irritation testing must be based on a thorough understanding of the mechanisms of ocular irritation. We have recently undertaken the task of developing such an understanding for a panel of surfactants. The purpose of this study was to expand our current panel of surfactants for which the microscopic changes occurring over time have been characterized. Macroscopic and microscopic findings regarding the ocular irritation of 6 surfactants of relatively unknown irritancy were compared to those of 6 surfactants of known irritancy. The right eye of each rat was treated by placing 10 μl of a surfactant directly on the cornea. Untreated left eyes served as the controls. At 3 hr and on days 1, 3, and 35, eyes and eyelids were collected for microscopic examination. Collectively, the macroscopic and microscopic findings revealed 3 surfactants to be similar to the mildly irritating surfactants previously studied, and 3 surfactants to be similar to the moderately irritating surfactant previously studied. Information such as this will be important to develop mechanistically based in vitro alternatives to replace the use of animals for ocular irritation testing.
Receptor and Nonreceptor-Mediated Organ-Specific Toxicity of Di(2-ethylhexyl)phthalate (DEHP) in Peroxisome Proliferator-Activated Receptorα-Null Mice
Authors: Ward Jerrold M. , Peters Jeffrey M. , Perella Christine M. , Gonzalez Frank J.
Published: September 2009

The peroxisome proliferator-activated receptora (PPARα) is the mediator of the biological effects of peroxisome proliferators through control of gene transcription. To determine if the toxic effects of di(2-ethylhexyl)phthalate (DEHP) are mediated by PPARα, we examined its effect in PPARα-null mice. Male Sv/129 mice, PPARα-null (–/–) or wild-type (+/+) were fed ad libitum either a control diet or one containing 12,000 ppm DEHP for up to 24 wk. Significant body weight loss and high mortality was observed in (+/+) mice fed DEHP. By 16 wk, all DEHP-fed (+/+) mice had died of cystic renal tubular disease. In contrast, the (–/–) mice fed DEHP had no changes in body weight until later in the study nor increased mortality. Histologically, (+/+) mice fed DEHP had typical toxic lesions in liver, kidney, and testis while (–/–) mice fed DEHP had no toxic liver lesions but did show evidence of toxicity in kidney and testis after 4–8 wk of feeding, which progressed into moderate lesions by 24 wk. Analysis of hepatic and renal mRNAs showed a typical pleiotropic response in gene expression in the DEHP-fed (+/+) mice that was absent in the DEHP-fed (–/ –) mice. These results provide evidence that PPARα mediates the subacute-chronic toxicity of DEHP in liver, kidney, and testis. However, because (-/–) mice did develop toxic lesions in kidney and testis, DEHP can also act through PPARα-independent pathways in mediating renal and testicular toxicity.
Immunohistochemical Detection of 8-Hydroxy-2'-deoxyguanosine in Paraffin-Embedded Sections of Rat Liver after Carbon Tetrachloride Treatment
Authors: Takahashi Satoru , Hirose Masao , Tamano Seiko , Ozaki Masakazu , Orita Shin-Ichiro , Ito Takayasu , Takeuchi Masao , Ochi Hirotomo , Fukada Shoko , Kasai Hiroshi , Shirai Tomoyuki
Published: September 2009

TO test the applicability of an anti-8-hydroxy-2'-deoxyguanosine (8-OH-dG) antibody for immunohistochemistry using paraffin-embedded sections, carbon tetrachloride (CCl4)-induced rat liver injury was evaluated. Male rats were given a single dose of CCl4 and killed at 6 hr, 12 hr, 1, 2, 3, and 7 days thereafter. Severe centrilobular necrosis was evident at 1 day. At 2 days, moderate mononuclear cell infiltration was present in centrilobular necrotic regions. Infiltrating mononuclear cells, surrounding sinusoidal endothelial cells and hepatocytes were stained with anti-8-OH-dG antibody at 2 and 3 days. Formation of 8-OH-dG in DNA and 8-oxo-dGTPase mRNA expression were also increased at these time points, the amounts of malondialdehyde and 4-hydroxy-2-nonenal showed 2 peaks at 6 hr and 3 days. The findings suggest that the main contributory factor in the massive hepatic necrosis was increased lipid peroxidation, rather than excessive formation of 8-OH-dG, and that the observed increase in the latter was largely due to infiltrating mononuclear cells. The agreement between biochemical data and the results for immunohistochemical analysis confirms that the anti-8-OH-dG antibody is applicable for detection of cells targeted by free radicals in paraffin-embedded sections and also for investigation of the mechanisms of oxidative damage-related disease, including carcinogenesis.
Effect of Phenobarbital on Hepatic Gap Junctional Intercellular Communication in Rats
Authors: Ito Seiichi , Tsuda Mina , Yoshitake Akira , Yanai Tokuma , Masegi Toshiaki
Published: September 2009

The effects of in vivo exposure to phenobarbital (PB) on hepatic gap junctional intercellular communication (GJIC) and conncxin protein expression in Sprague-Dawley rats were examined by in vivo/in vitro dye-transfer assay, immunohistochemical staining, and by Western blot analysis. PB (50 mg/kg) was administered orally once a day for up to 6 wk. The average size of the dye spread after injection of Lucifer Yellow decreased at week 1 and remained at the same level until week 6. The area and number of connexin 32 (Cx32) spots per hepatocyte in the central zone of liver lobules decreased from week 1 to week 6, but no change of Cx32 spots in the peripheral zone was observed. The average area and number of connexin 26 (Cx26) spots per hepatocytes showed no clear change through the experimental periods. The decreased level of Cx32 protein in plasma membranes was observed in the PB group. These results suggest that PB, a liver tumor-promoting agent, inhibits hepatic GJIC in vivo in rats and that aberrant Cx32 protein expression and/or localization may be responsible for this effect.
Brevetoxicosis in Manatees(Trichechus manatus latirostris)from the 1996 Epizootic: Gross, Histologic, and Immunohistochemical Features
Authors: Bossart Gregory D. , Baden Daniel G. , Ewing Ruth Y. , Roberts Brenda , Wright Scott D.
Published: September 2009

In 1996, at least 149 manatees (Trichechus manatus latirostris) died in an unprecedented epizootic along the southwest coast of Florida. At about the same time, a bloom of the brevetoxin-producing dinoflagellates, Gymnodinium breve, was present in the same area. Grossly, severe nasopharyngeal, pulmonary, hepatic, renal, and cerebral congestion was present in all cases. Nasopharyngeal and pulmonary edema and hemorrhage were also seen. Consistent microscopic lesions consisted of catarrhal rhinitis nulm hemorrhage and edema, multiorgan hemosiderosis, and nonsuppurative leptomeningitis. Immunohistochemical staining using a polyclonal primary antibody to brevetoxin (GAB) showed intense positive staining of lymphocytes and macrophages in the lung liver and secondary lymphoid tissues. Additionally, lymphocytes and macrophages associated with the inflammatory lesions of the nasal mucosa and meninges were also positive for brevetoxin. These findings implicate brevetoxicosis as a component of and the likelv primary etiology for the epizootic. The data suggest that mortality resulting from brevetoxicosis may not necessarily be acute but may occur after chronic inhalation and/or ingestion. Immunohistochemical staining with interleukin-1-β-converting enzyme showed positive staining with a cellular tropism similar to GAB. This suggests that brevetoxicosis may initiate apoptosis and/or the release of inflammatory mediators that culminate in fatal toxic shock.
Spontaneous Cystic Keratinizing Epithelioma in the Lung of a Sprague-Dawley Rat
Authors: Rittinghausen S. , Kaspareit J.
Published: September 2009

A spontaneous cystic keratinizing epithelioma in the lung of a 704-day-old female Hsd: Sprague-Dawley rat is described. The rat had a white subpleural discoloration on 1 diaphragmatic lobe at necropsy. Microscopically, a central keratin mass, surrounded by a multilayered squamous epithelium of varying thickness, was found. In the periphery of the tumor, epithelial nests projected multifocally into the adjacent alveolar spaces. Immunohistochemical labeling of nuclei by PCNA (proliferating cell nuclear antigen) revealed proliferative activity in 1 or 2 peripheral cell layers in most locations. To the best of the authors' knowledge, this is the first report dealing with the morphological and immunohistochemical features of a spontaneous cystic keratinizing epithelioma in a rat.
A Review of Chronic Inhalation Studies with Mainstream Cigarett Smoke in Rats and Mice
Authors: Coggins Christopher R. E.
Published: September 2009

In this paper, I review the results of a representative selection of chronic inhalation studies with rats and mice exposed to mainstream cigarette smoke and describe the inhalation exposures and the histopathological changes reported by various authors. Many of the studies used nose-only exposure systems, whereas others simply used large whole-body chambers. Smoke-induced epithelial hypertrophy, hyperplasia, and squamous metaplasia were reported in the conducting airways in most of the studies, along with increased numbers of intra-alveolar macrophages that were occasionally associated with alveolar metaplasia. Lung adenomas and adenocarcinomas were reported in only a few of the studies. No statistically significant increase in the incidence of malignant lung tumors was seen in either species as a result of smoke exposure, a finding that does not agree with the results of epidemiological studies in humans. Possible reasons for this lack of correlation are given.
Cytokeratin Expression Patterns in the Rat Respiratory Tract as Markers of Epithelial Differentiation in Inhalation Toxicology. I. Determination of Normal Cytokeratin Expression Patterns in Nose, Larynx, Trachea, and Lung
Authors: Schlage Walter K. , Bülles Heidi , Friedrichs Detlef , Kuhn Maria , Teredesai Ashok
Published: September 2009

Cytokeratin (CK) polypeptides constitute the intermediate filament cytoskeleton of epithelial cells. The patterns of CK expression can be regarded as specific markers for the epithelial differentiation status. Our objective was to map the cell type-specific CK expression patterns at all representative sites of the respiratory tract of untreated rats to use as a base for the detection of inhalation exposure-related differentiation changes. Using routine paraffin-embedded sections and a panel of well-characterized monoclonal antibodies for immunohistochemistry, we obtained CK staining patterns as follows. Nasal cavity: respiratory epithelium CKI8, CK19 (basal, ciliated, nonciliated cells), CK14, and/or CK15 (basal and nonciliated cells); olfactory epithelium CK18 (basal, mid, apical zones and Bowman's glands), CK14, and CK15 (basal zone); squamous epithelium of ventral meatus CK14, CK15 (basal and supra-basal cells), CK1, 10/11, and CK13 (suprabasal cells); glands and columnar epithelia of vomeronasal organ and nasolacrimal duct CK7 and CK13 in addition to respiratory epithelial CK pattern. Trachea: similar to nasal respiratory epithelium with pronounced CK15 and additional CK7. Larynx: CK14, CK15 (basal, ciliated, nonciliated cells), CK8, CK18, CK19 (not in basal cells), CK4, and CK13 (cuboidal and squamoid cells of ventral half). Lung: bronchial epithelium CK14 and CK15 (basal cells only); bronchial and alveolar epithelium CK7, CK8, CK18, and CK19; bronchiolar epithelium similar but less CK8 and no CK7; pleural mesothelium CK7, CK8, and CK19. This inventory of complex CK expression patterns provides the basis for investigating test substance-related effects in inhalation toxicology, e.g., cigarette smoke-induced changes.
Effect of Hexachloro-1,3-butadiene on Renal Carcinogenesis in Male Rats Pretreated with N-Ethyl-N-hydroxyethylnitrosamine
Authors: Nakagawa Yoshinori , Kitahori Yoshiteru , Cho Masaki , Konishi Noboru , Tsumatani Ken-Ichi , Ozono Seiichiro , Okajima Eigoro , Hirao Yoshihiko , Hiasa Yoshio
Published: September 2009

Hexachloro-1,3-butadiene (HCBD) is a potent nephrotoxicant that selectively damages the straight portion (pars recta) of the proximal tubule in the rat. To determine its effects on carcinogenesis, HCBD was administered for 30 wk at a concentration of 0.1% by weight in basal diet to male Wistar rats previously given 0.1% Af-ethyl-W-hydroxyethylnitrosamine (EHEN) in the drinking water for 2 wk. The combined treatment resulted in a significantly higher incidence of renal cell tumors than when EHEN was administered alone. This chronic exposure and a short course of a 0.2% HCBD diet for 3 wk caused marked increase in the numbers of bromo-deoxyuridine-incorporating cells or proliferating cell nuclear antigen-positive cells in the outer stripe of the kidney. The ability of HCBD to promote EHEN-initiated renal tumorigenesis in rats thus appears to be associated intimately with linked nephropathy and subsequent cell proliferation.
Predominant p53 G→A Transition Mutation and Enhanced Cell Proliferation in Uterine Sarcomas of CBA Mice Treated with 1,2-Dimethylhydrazine
Authors: Trukhanova Lyuba S. , Hong Hue-Hua L. , Sills Robert C. , Bowser Alicia D. , Gaul Beth , Boorman Gary A. , Turusov Vladimir S. , Devereux Theodora R. , Dixon Darlene
Published: September 2009

Mouse uterine tumors were examined for genetic alterations in the ras proto-oncogene and p53 tumor suppressor gene arid for other biologically relevant immunohistochemical markers that may increase our understanding of the events that occur in uterine cancer. Fourteen dimethylhydrazine (DMH)-induced uterine sarcomas, including 3 primary malignant fibrous histiocytomas (MFH), 7 transplanted MFH, 3 stromal sarcomas, and 1 undifferentiated sarcoma, were first screened by immunohistochemistry for p53 missense mutations, followed by single strand conformation polymorphism analysis and DNA sequencing for the identification of point mutations. There was 100% correlation between p53 protein immunopositivity and subsequent detection ofp53 mutations in DMH-induced malignant fibrous histiocytomas. All MFH had a characteristic p53 G:C→A:T transition mutation, consistent with O6-methylguanine mispairing with thymine, the most common DNA lesion caused by alkylating agents. DMH-induced uterine MFH with p53 mutations also had a higher proliferative rate (qualitatively evaluated by immunohistochemical detection of proliferating cell nuclear antigen) when compared with other DMH-induced sarcomas. Uterine sarcomas were further evaluated for biological end points, such as estrogen receptor and desmin. Neoplastic cells from stromal sarcomas (SS), undifferentiated sarcomas (US), and MFH did not stain for desmin. The estrogen receptor was detected in normal uteri and a small portion of MFH, SS, and US. Our data suggest that DMH-induced uterine sarcomas are not consistent with smooth muscle cell origin and that a subset of these tumors, specifically DMH-induced malignant fibrous histiocytomas, have unique p53 G:C→A:T transitions and a high proliferative rate.
Arteriopathy Induced by an Adenosine Agonist-Antihypertensive in Monkeys
Authors: Albassam Mudher A. , Smith Graham S. , Macallum Grace E.
Published: September 2009

An adenosine agonist, designated chemically as (R)-N-(2,3-dihydro-lH-inden-1-yl) adenosine or CI-947, was administered orally to 2 males and 2 female cynomolgus monkeys each at 5, 10, 20, and 50 mg/kg of body weight for 2 wk. One male and 1 female given 50 mg/kg were euthanatized on days 10 and 8, respectively, because of poor clinical condition. Emesis was present at 10, 20, and 50 mg/kg. Decreased heart rate and QT prolongation were present at 50 mg/kg. Extramural coronary arterial lesions consisting of medial necrosis with cellular debris and mixed inflammatory cell response in the intima, media, and adventitia were present in 1 male at 20 mg/kg and 1 male at 50 mg/kg at study termination. Similar arterial lesions were present in the small and large intestines and testis of the male at 50 mg/kg. Colonic mucosal erosions with mixed inflammatory cell infiltrates in the lamina propria were seen in this male and in all CI-947 treated females at 10, 20, and 50 mg/kg. Myocardial degeneration and necrosis of myocardial fibers with mononuclear cell infiltrates in the interstitium were noted in the left ventricle of 1 female at 20 mg/kg and in all animals at 50 mg/kg. Renal cortical tubular dilatation with increases in serum creatinine and/or blood urea nitrogen were noted in a control female and animals at 10 and 50 mg/kg. Plasma CI-947 concentration increased with increasing dose. Coronary vascular injury in the monkey was similar to the arterial lesion in CI-947-treated dogs and may relate to the pharmacologic/hemodynamic effects induced by CI-947. When compared with the dog, the monkey appears to be less sensitive to development of arteriopathy, as indicated by lower incidence, at similar systemic exposure levels.
Metal Storage and Transport Proteins Increase After Exposure of the Rat Lung to an Air Pollution Particle
Authors: Ghio Andrew J. , Richards Judy H. , Dittrich Kathleen L. , Samet James M.
Published: September 2009

With the single exception of mercury, all metals in the atmosphere are associated with particles. The lungs are subsequently exposed to metals present in air pollution particles on a continuous basis. Because metal exposure can be associated with an oxidative stress, a mechanism that isolates the metal in a chemically less reactive form would be of benefit. We tested the hypothesis that the concentrations of both lactoferrin and ferritin in the rat lung increase after exposure to a metal-rich emission source air pollution particle. Using immunohistochemistry, we characterized changes in the concentrations of ferritin and lactoferrin after exposure of rats to an emission source air pollution particle. Lavage metal concentrations, measured by inductively coupled plasma emission spectroscopy, increased 4 hr after exposure to an oil fly ash. After exposure to this metal-rich emission source air pollution particle, ferritin concentrations in the lower respiratory tract increased. Comparable to the iron-storage protein, concentrations of both lactoferrin and transferrin were elevated after exposure. The greatest concentrations of ferritin, lactoferrin, and transferrin occurred at approximately 24 hr after exposure to the air pollution particle. Levels then decreased, and by 96 hr after instillation of the oil fly ash, ferritin, lactoferrin, and transferrin were not elevated relative to those animals exposed to saline. We conclude that, in response to an emission source air pollution particle with high concentrations of metals, there is an increase in ferritin, lactoferrin, and transferrin concentrations in the lungs of the host. The function of these increases in iron-binding proteins may be to control the oxidative stress associated with the exposure to metals.
Immunohistochemical Localization of Microcystin-LR in the Liver of Mice: A Study on the Pathogenesis of Microcystin-LR-Induced Hepatotoxicity
Authors: Yoshida Toshinori , Makita Yuki , Tsutsumi Tomoaki , Nagata Satoshi , Tashiro Fumio , Yoshida Fuyuko , Sekuima Masaru , Tamura Shin-Ichi , Harada Takanori , Maita Keizo , Ueno Yoshio
Published: September 2009

The relationship between the intralobular sites of hepatotoxic injury and the distribution of microcystin-LR (MCLR), an inhibitor of protein phosphatases 1 (PP1) and 2A (PP2A), was examined using an immunohistochemical method with a monoclonal antibody specific to MCLR on the livers of mice receiving a single ip injection of the MCLR. Immunoblotting and high-performance liquid chromatography analyses of liver extracts were also performed to determine the binding form of MCLR to PP1 and PP2A (MCLR-PP1/PP2A adducts) and free MCLR. Immunohistochemistry revealed a discernible intensity of staining in the centrilobular regions where hemorrhage and apoptosis occurred. In these regions, immunopositivity was evident in the cytoplasm and nuclei of the hepa-tocytes; some apoptotic cells were also immunopositive. In contrast, coaglilative necrosis, which was mainly'evident in the midlobular regions, was completely negative. Analysis of liver extracts demonstrated MCLR-PP1/PP2A adducts, but free MCLR was below detection limit. These results suggest that the immunohistochemical localization of MCLR in centrilobular hepatocytes is closely associated with the onset of hemorrhage and apoptosis and is related to adduct formation. The occurrence of coagulative necrosis however might also be related to other factors such as ischemia/hypoxia.
Spontaneous Neoplasm Incidences in Fischer 344 Rats and B6C3F1 Mice in Two-Year Carcinogenicity Studies: A National Toxicology Program Update
Authors: Haseman Joseph K. , Hailey James R. , Morris Richard W.
Published: September 2009

Spontaneous neoplasm rates were determined for control Fischer 344 (F344) rats and B6C3F, mice from 2-yr rodent carcinogenicity studies carried out by the National Toxicology Program (NTP). The most frequently occurring neoplasms in untreated male F344 rats were testicular adenoma (89.1%), mononuclear cell leukemia (50.5%), adrenal gland pheochromocytoma (31.9%), and pituitary gland neoplasms (30.4%). For untreated female F344 rats, the most frequently occurring neoplasms were pituitary gland neoplasms (54.2%), mammary gland fibroadenoma (41.2%), and mononuclear cell leukemia (28.1%). The most frequently occurring neoplasms in untreated male B6C3F, mice were liver adenoma/carcinoma (42.2%), lung adenoma/carcinoma (20.5%), and malignant lymphoma (8.3%). For untreated female B6C3F, mice, the most frequently occurring neoplasms were liver adenoma/carcinoma (23.6%), malignant lymphoma (20.9%), and pituitary gland adenoma/carcinoma (1.4.8%). The tumor rates observed in feeding study (untreated) and inhalation study (chamber) control rats were generally similar. The major exceptions were pituitary gland tumors and testicular adenoma in male F344 rats. The overall incidence of testicular adenoma was much lower in chamber controls (69.4%) than in feeding study controls (89.1%), whereas pituitary gland neoplasms showed the opposite trend (60.7% vs 30.4%). The most likely explanation for this difference is related to the individual housing of chamber controls and the group housing of feeding study controls. Differences in diagnostic criteria may influence reported tumor rates. To ensure consistency and comparability of tumor diagnosis from study to study, the NTP uses rigorous histopathology quality assurance and peer review procedures. Biological factors such as body weight may also affect tumor incidence. For example, increased body weights are associated with increased incidences of certain site-specific neoplasms, especially pituitary gland and mammary gland neoplasms in rats and liver tumors in mice. The presence of Helicobacter hepaticus has been associated with an increased incidence of liver neoplasms in male B6C3F, mice. Other factors that may produce differences in control tumor rates from study to study include diet, environmental factors, genetic drift, study duration, and survival differences. The NTP database provides historical control data that may be useful in the evaluation of possible chemically related changes in tumor incidence. However, it is essential that the study being evaluated be comparable to those in the NTP database with respect to those factors that are known to influence tumor occurrence.
Tripropylene Glycol Diacrylate but Not Ethyl Acrylate Induces Skin Tumors in a Twenty-Week Short-Term Tumorigenesis Study in Tg.AC (v-Ha-ras) Mice
Authors: Nylander-French Leena A. , French John E.
Published: September 2009

The toxicity of the esters of acrylic acid are poorly understood even though significant human exposure occurs. To conduct rapid comparative short-term bioassays, we used the Tg.AC (v-Ha- ras) transgenic mouse model to determine the toxicity and potential carcinogenicity of tripropylene glycol diacrylate (TPGDA) alone and in a reference formulated ultraviolet radiation curable lacquer (Lacquer A), which is used in the ultraviolet radiation curable surface coatings. For comparison, ethyl acrylate (EA) was used as a reference acrylate. Insertion of the ζ-globin promoted v-Ha- ras transgene into the FVB mouse genome (Tg.AC) introduced a defined genetic lesion, which is critical but insufficient by itself to induce benign or malignant tumors in the skin unless activated. Activation and expression of the transgenic ras oncoprotein in this mouse line induces a dose-related increase in papillomas (skin reporter phenotype) within weeks. Based on dose-related increases in skin hyperplasia following dermal exposure to EA, TPGDA, or Lacquer A (applied equimolar for TPGDA concentration), the dosing regimen was selected. Starting at 12 wk of age, the agents were administered topically (200 μl of acetone vehicle) 3 times/vk for 20 wk to the shaved dorsal skin of female Tg.AC mice (n = 10/group). TPGDA and reference Lacquer A (equimolar for TPGDA) at 5 or 10 μmoles/mouse but not EA (60, 300, or 600 (μmoles/mouse) or TPGDA or Lacquer A at 1 μmole/mouse induced a dose-related increase in papillomas between 6 and 12 wk of treatment that reached a maximum number of papillomas per mouse between 19 and 20 wk of treatment. These results indicate that TPGDA is significantly more potent than EA for inducing the skin reporter phenotype and may be predicted to be carcinogenic in long-term cancer bioassays at the site of contact.
Carcinogenic Responses of Transgenic Heterozygous p53 Knockout Mice to Inhaled 239PuO2 or Metallic Beryllium
Authors: Finch Gregory L. , March Thomas H. , Hahn Fletcher E. , Barr Edward B. , Belinsky Steven A. , Hoover Mark D. , Lechner John E. , Nikula Kristen J. , Hobbs Charles H.
Published: September 2009

The transgenic heterozygous p53+/- knockout mouse has been a model for assessing the tumorigenicity of selected carcinogens administered by noninhalation routes of exposure. The sensitivity of the model for predicting cancer by inhaled chemicals has not been examined. This study addresses this issue by acutely exposing p53+/- mice of both sexes by nose-only inhalation to either air (controls), or to 1 of 2 levels of 239PuO2 (500 or 100 Bq 239Pu) or beryllium (Be) metal (60 or 15 μg). Additional wild-type p53+/- mice were exposed by inhalation to either 500 Bq of 239PuO2 or 60 μg of Be metal. These carcinogens were selected because they operate by differing mechanisms and because of their use in other pulmonary carcinogenesis studies in our laboratory. Four or 5 of the 15 mice per sex from each group were sacrificed 6 mo after exposure, and only 2 pulmonary neoplasms were observed. The remainder of the mice were held for life-span observation and euthanasia as they became moribund. Survival of the p53+/- knockout mice was reduced compared to the p53+/+ wild-type mice. No lung neoplasms were observed in p53+/- mice exposed to air alone. Eleven of the p53+/- mice inhaling 239PuO2 developed pulmonary neoplasms. Seven p53+/+ mice exposed to 239PuO2 also developed pulmonary neoplasms, but the latency period for pulmonary neoplasia was significantly shorter in the p53+/- mice. Four pulmonary neoplasms were observed in p53+/- mice exposed to the higher dose of Be, whereas none were observed in the wild-type mice or in the heterozygous mice exposed to the lower dose of Be. Thus, both p53+/- and p53+/+ mice were susceptible to 239Pu-induced carcinogenesis, whereas the 53+/- but not the p53+/+ mice were susceptible to Be-induced carcinogenesis. However, only 2 pulmonary neoplasms (1 in each of the 239PuO2 exposure groups) were observed in the 59 p53+/- mice that were sacrificed or euthanatized within 9 mo after exposure, indicating that the p53+/- knockout mouse might not be appropriate for a 6-mo model of carcinogenesis for these inhaled carcinogens.
Phenobarbital Does Not Promote Hepatic Tumorigenesis in a Twenty-Six-Week Bioassay inp53Heterozygous Mice
Authors: Sagartz John E. , Curtiss Sandra W. , Bunch Roderick T. , Davila Julio C. , Morris Dale L. , Alden Carl L.
Published: September 2009

The tumorigenic potential of phenobarbital was examined in a 26-wk carcinogenesis bioassay using p53 heterozygous mice and wild-type controls. Fifteen mice/sex/genotype were exposed to either 500 or 1,000 ppm phenobarbital in the diet. Dietary administration of 3,750 ppm p-cresidine, a transspecies mutagenic carcinogen, to both heterozygous and wild-type mice served as a positive control. Phenobarbital treatment caused increases in liver: body weight ratios and histologic evidence of centrilobular hepatocellular hypertrophy. No tumors were observed in any phenobarbital-treated mice. Mice given p-cresidine exhibited a moderate reduction in body weight gain over the couise of the study. Heterozygous mice treated with p-cresidine exhibited a high incidence of urinary bladder tumors. Similar tumors were also present in a small number of p-cresidine-treated wild-type mice. Our results demonstrate the lack of a hepatic tumor response to phenobarbital, a compound that is a potent and prototypic hepatic microsomal enzyme inducer, a nongenotoxic rodent carcinogen, and a human noncarcinogen. This finding supports the continued utility of this model as an alternative to the mouse bioassay for human carcinogenic safety assessment of potentially genotoxic carcinogens because it did not produce a false-positive response to this potent nongenotoxic agent.