Showing papers in "Toxicologic Pathology in 2004"

Brain inflammation and Alzheimer's-like pathology in individuals exposed to severe air pollution.

Abstract: Air pollution is a complex mixture of gases (e.g., ozone), particulate matter, and organic compounds present in outdoor and indoor air. Dogs exposed to severe air pollution exhibit chronic inflammation and acceleration of Alzheimer's-like pathology, suggesting that the brain is adversely affected by pollutants. We investigated whether residency in cities with high levels of air pollution is associated with human brain inflammation. Expression of cyclooxygenase-2 (COX2), an inflammatory mediator, and accumulation of the 42-amino acid form of beta-amyloid (Abeta42), a cause of neuronal dysfunction, were measured in autopsy brain tissues of cognitively and neurologically intact lifelong residents of cities having low (n:9) or high (n:10) levels of air pollution. Genomic DNA apurinic/apyrimidinic sites, nuclear factor-kappaB activation and apolipoprotein E genotype were also evaluated. Residents of cities with severe air pollution had significantly higher COX2 expression in frontal cortex and hippocampus and greater neuronal and astrocytic accumulation of Abeta42 compared to residents in low air pollution cities. Increased COX2 expression and Abeta42 accumulation were also observed in the olfactory bulb. These findings suggest that exposure to severe air pollution is associated with brain inflammation and Abeta42 accumulation, two causes of neuronal dysfunction that precede the appearance of neuritic plaques and neurofibrillary tangles, hallmarks of Alzheimer's disease.

Structure-Based Thresholds of Toxicological Concern (TTC): Guidance for Application to Substances Present at Low Levels in the Diet

Abstract: The threshold of toxicological concern (TTC) is a pragmatic risk assessment tool that is based on the principle of establishing a human exposure threshold value for all chemicals, below which there is a very low probability of an appreciable risk to human health. The concept that there are levels of exposure that do not cause adverse effects is inherent in setting acceptable daily intakes (ADIs) for chemicals with known toxicological profiles. The TTC principle extends this concept by proposing that a de minimis value can be identified for many chemicals, in the absence of a full toxicity database, based on their chemical structures and the known toxicity of chemicals which share similar structural characteristics. The establishment and application of widely accepted TTC values would benefit consumers, industry and regulators. By avoiding unnecessary toxicity testing and safety evaluations when human intakes are below such a threshold, application of the TTC approach would focus limited resources of time, cost, animal use and expertise on the testing and evaluation of substances with the greatest potential to pose risks to human health and thereby contribute to a reduction in the use of animals. An Expert Group of the European branch of the International Life Sciences Institute-ILSI Europe-has examined the TTC principle for its wider applicability in food safety evaluation. The Expert Group examined metabolism and accumulation, structural alerts, endocrine disrupting chemicals and specific endpoints, such as neurotoxicity, teratogenicity, developmental toxicity, allergenicity and immunotoxicity, and determined whether such properties or endpoints had to be taken into consideration specifically in a step-wise approach. The Expert Group concluded that the TTC principle can be applied for low concentrations in food of chemicals that lack toxicity data, provided that there is a sound intake estimate. The use of a decision tree to apply the TTC principle is proposed, and this paper describes the step-wise process in detail. Proteins, heavy metals and polyhalogenated-dibenzodioxins and related compounds were excluded from this approach. When assessing a chemical, a review of prior knowledge and context of use should always precede the use of the TTC decision tree. The initial step is the identification and evaluation of possible genotoxic and/or high potency carcinogens. Following this step, non-genotoxic substances are evaluated in a sequence of steps related to the concerns that would be associated with increasing intakes. For organophosphates a TTC of 18microg per person per day (0.3 microg/kg bw/day) is proposed, and when the compound is not an OP, the TTC values for the Cramer structural classes III, II and I, with their respective TTC levels (e.g. 1800, 540 and 90 microg per person per day; or 30, 9 and 1.5 microg/kg bw /day), would be applied sequentially. All other endpoints or properties were shown to have a distribution of no observed effect levels (NOELs) similar to the distribution of NOELs for general toxicity endpoints in Cramer classes I, II and III. The document was discussed with a wider audience during a workshop held in March 2003 (see list of workshop participants).

Relationships Between Organ Weight and Body/Brain Weight in the Rat: What Is the Best Analytical Endpoint?:

Abstract: Analysis of organ weight in toxicology studies is an important endpoint for identification of potentially harmful effects of chemicals. Differences in organ weight between treatment groups are often accompanied by differences in body weight between these groups, making interpretation of organ weight differences more difficult. Using data from control rats that were part of 26 toxicity studies conducted under similar conditions, we have evaluated the relationship between organ weight and body/brain weight to determine which endpoint (organ weight, organ-to-body weight ratio, or organ-to-brain weight ratio) is likely to accurately detect target organ toxicity. This evaluation has shown that analysis of organ-to-body weight ratios is predictive for evaluating liver and thyroid gland weights, and organ-to-brain weight ratios is predictive for evaluating ovary and adrenal gland weights. Brain, heart, kidney, pituitary gland, and testes weights are not modeled well by any of the choices, and alternative analysis methods such as analysis of covariance should be utilized.

Bone Neoplasms in F344 Rats Given Teriparatide [rhPTH(1-34)] Are Dependent on Duration of Treatment and Dose

Abstract: A long-term study was conducted in female F344 rats to determine the relative importance of dose, treatment duration, and age at initiation of treatment on the incidence of teriparatide [rhPTH[1-34)]-induced bone proliferative lesions. Treatment groups consisted of different combinations of dose (0, 5, or 30 μg/kg/d), treatment duration (6, 20, or 24 months) and age at initiation of treatment (2 or 6 months of age). The primary endpoints were the incidence of bone neoplasms and effects on bone mass and structure as evaluated by quantitative computed tomography and histomorphometery. Significant increases in the incidence of bone tumors (osteoma, osteoblastoma, and osteosarcoma) occurred in rats treated with 30 μg/kg for 20 or 24 months. No neoplasms were found when the 5 μg/kg treatment was initiated at 6 months of age and continued for either 6 or 20 months (up to 70% of life span). This treatment regimen defined a “no-effect” dose for neoplasm formation that nevertheless resulted in substantial increase...

Best Practices Guideline: Toxicologic Histopathology

Abstract: Health & Environmental Sciences, Dow Corning Corporation, Midland, Michigan 48686 Covance Laboratories Inc., Vienna, Virginia 22182 Pathco Inc., Frederick, Maryland 21702 Laboratory of Experimental Pathology, National Institute of Environmental Health, Research Triangle Park, North Carolina 27709 Center for Veterinary Medicine, Food and Drug Administration, Rockville, Maryland 20855 Estuary Pharmaceuticals, Cambridge, Massachusetts 02139 Pfizer Inc., Kalamazoo, Michigan 49001 and Chesterfield, Missouri 63005

Toxicoproteomics: Proteomics applied to toxicology and pathology

Abstract: Global measurement of proteins and their many attributes in tissues and biofluids defines the field of proteomics. Toxicoproteomics, as part of the larger field of toxicogenomics. seeks to identify critical proteins and pathways in biological systems that are affected by and respond to adverse chemical and environmental exposures using global protein expression technologies. Toxicoproteomics integrates 3 disciplinary areas: traditional toxicology and pathology, differential protein and gene expression analysis, and systems biology. Key topics to be reviewed are the evolution of proteomics, proteomic technology platforms and their capabilities with exemplary studies from biology and medicine, a review of over 50 recent studies applying proteomic analysis to toxicological research, and the recent development of databases designed to integrate -Omics technologies with toxicology and pathology. Proteomics is examined for its potential in discovery of new biomarkers and toxicity signatures, in mapping serum,plasma. and other biofluid proteomes, and in parallel proteomic and transcriptomic studies. The new field of toxicoproteomics is uniquely positioned toward an expanded understanding of protein expression during toxicity and environmental disease for the advancement of public health.

Serum Troponins as Biomarkers of Drug-Induced Cardiac Toxicity

Abstract: Member of the Expert Working Group and Chair of the Expert Working Group and Corresponding Author, Professor, Department of Biochemistry & Molecular Biology, University of Minnesota School of Medicine, Duluth, MN FDA Liaison and FDA Center for Drug Evaluation and Research, Rockville, MD 20852 Center for Drug Evaluation and Research, FDA, Laurel, MD 20708 Principal Scientist, Oxford GlycoSciences, Montgomery Village, MD 20886-1265 FDA National Center for Toxicological Research, Rockville, MD 20857 Drug Safety Evaluation, Global Research and Development, Ann Arbor Laboratories, Pfizer Inc., Ann Arbor, MI 48105 Laboratory of Molecular Carcinogenesis, National Institutes of Environmental Health Sciences, Research Triangle Park, NC 27709 Director, General Toxicology, Drug Safety and Metabolism, Schering-Plough Research Institute, Lafayette, NJ 07848, and Manager, Clinical Pathology Laboratory, Preclinical Safety Sciences, GlaxoSmithKline, Hertfordshire, SG12, ODP, United Kingdom

Invited Review: A Contemporary Overview of Chronic Progressive Nephropathy in the Laboratory Rat, and Its Significance for Human Risk Assessment:

Abstract: CPN (chronic progressive nephropathy) is a spontaneous age-related disease that occurs in high incidence in the strains of rat commonly used in preclinical toxicology studies, exhibiting a male predisposition. Although increasing in incidence and severity with age, evidence indicates that CPN should be regarded as a specific disease entity and not just a manifestation of the aging process. A number of factors, mainly dietary manipulations, have been shown to modify the expression of CPN. Amongst these, restriction of caloric intake is the most effective for inhibiting the disease process. The precise etiology of CPN and the mechanism(s) underlying its pathogenesis remain unknown, but the long-standing assumption that glomerular dysfunction is the primary basis is challenged in the light of contemporary developments in understanding filtration and postglomerular cellular processing of albumin. CPN is not only a degenerative disease, but also has regenerative aspects with a high cell proliferative rate in affected tubules. Accordingly, evidence is emerging that advanced, particularly end-stage CPN, is a risk factor for a marginal increase in the background incidence of renal tubule tumors. Many chemicals are known to exacerbate the severity of CPN to an advanced stage, and this interaction between chemical and CPN can result in a small increase in the incidence of renal adenomas in 2-year carcinogenicity bioassays. Review of the pathological entities associated with chronic renal failure in man emphasizes that this rodent condition has no strict human counterpart. Because CPN is a rodent-specific entity, the finding of a small, statistically significant increase in renal tubule tumors, linked to exacerbation of CPN by a test chemical in a preclinical study for carcinogenicity, can be regarded as having no relevance for extrapolation in human risk assessment.

Kidney slices of human and rat to characterize cisplatin-induced injury on cellular pathways and morphology.

Abstract: Kidney slices represent an in vitro model that has the cellular complexity of in vivo tissue to provide insights into mechanisms of organ injury, as shown in this study with the model nephrotoxicant cisplatin. Cell pathways altered by cisplatin exposure are assessed by gene expression analysis, cell function, and morphology in human and rat kidney slices in comparison to rat kidney from an in vivo study. The acute nephrosis of the tubular epithelium induced by cisplatin in vivo was reproduced in both human and rat kidney slices, while the glomerulus appeared resistant even at high concentrations. Kidney gene expression changes of in vivo and in vitro samples were indicative of transcription, DNA damage, cell cycle, proliferation, and apoptosis that are in agreement with the mechanism of cisplatin causing DNA damage, growth arrest, and apoptosis; while genes indicative of protein damage, the disruption of transport and calcium homeostasis, cellular metabolism, and oxidative stress are pathways linked with cisplatin binding to various cellular proteins and macromolecules. Both concentration and time-dependent gene expression changes evident in the in vitro model preceded a change in tissue morphology. Functional assays confirming cell dysfunction and increased apoptosis revealed the rat kidney to be more sensitive to the effects of cisplatin than human kidney as demonstrated by significant decreases in slice ATP and GSH levels, significant increases in caspase 9 and 3 activity, p53 protein levels, and increased DNA laddering. The regional markers of proximal and distal tubular injury, alpha- and pi-glutathione S-transferases, were shown for the human kidney slices to be significantly increased by cisplatin. In this study, cisplatin-induced nephrotoxicity was demonstrated morphologically in rat and human kidney slices, and the associated gene expression and functional changes characterized the cellular pathways involved.

Male reproductive tract lesions at 6, 12, and 18 months of age following in utero exposure to di(n-butyl) phthalate

Abstract: In utero exposure of male rats to the antiandrogen di(n-butyl) phthalate (DBP) leads to decreased anogenital distance (AGD) on postnatal day (PND) 1, increased areolae retention on PND 13, malformations in the male reproductive tract, and histologic testicular lesions including marked seminiferous epithelial degeneration and a low incidence of Leydig cell (LC) adenomas on PND 90. One objective of this study was to determine the incidence and persistence of decreased AGD, increased areolae retention, and LC adenomas in adult rats following in utero DBP exposure. A second objective was to determine whether AGD and areolae retention during the early postnatal period are associated with lesions in the male reproductive tract. Pregnant Crl:CD(SD)BR rats were gavaged with corn oil or DBP at 100 or 500 mg/kg/day, 10 dams per group. Three replicates of rats (n = 30 rats per replicate) were exposed from gestation day 12 to 21 and the male offspring allowed to mature to 6, 12, or 18 months of age. Gross malformations in the male reproductive tract and histologic lesions in the testes were similar to those previously described. However, testicular dysgenesis, a lesion of proliferating LCs and aberrant tubules that has not been previously described in DBP-exposed testes, was diagnosed. The incidence of this lesion was approximately 20% unilateral and 7‐18% bilateral in the high-dose group and was similar among all ages examined, implicating a developmental alteration rather than an age-related change. AGD and areolae retention were found to be permanent changes following in utero exposure to 500 mg/kg/day of DBP. Decreased AGD was a sensitive predictor of lesions in the male reproductive tract, relatively small changes in AGD were associated with a significant incidence of male reproductive malformations. In utero DBP exposure induced proliferative developmental lesions, some of which would have been diagnosed as LC adenomas by the morphological criteria set forth by the Society of Toxicologic Pathology. However, these lesions were dissimilar to traditional LC adenomas as the LCs were poorly differentiated and the lesions contained aberrant seminiferous tubules. While the morphology and incidence of this DBP-induced testicular developmental lesion has been fully characterized by this study, the detailed pathogenesis warrants further investigation.

Prediction of rodent carcinogenesis: an evaluation of prechronic liver lesions as forecasters of liver tumors in NTP carcinogenicity studies.

Abstract: The National Toxicology Program (NTP) developed the chronic 2-year bioassay as a mechanism for predicting the carcinogenic potential of chemicals in humans. The cost and duration of these studies has limited their use to small numbers of selected chemicals. Many different short-term methods aimed at increasing predictive accuracy and the number of chemicals evaluated have been developed in attempts to successfully correlate their results with evidence of carcinogenicity (or lack of carcinogenicity) are assessed. Using NTP studies, the effectiveness of correlating prechronic liver lesions with liver cancer encompassing multiple studies using mice (83 compounds) and rats (87 compounds). These lesions include hepatocellular necrosis, hepatocellular hypertrophy, hepatocellular cytomegaly, bile duct hyperplasia, and hepatocellular degeneration, along with increased liver weight. Our results indicate that pooling 3 of these prechronic data points (hepatocellular necrosis, hepatocellular hypertrophy, and hepatocellular cytomegaly) can be very predictive of carcinogenicity in the 2-year study (p < 0.05). The inclusion of increased liver weight as an endpoint in the pool of data points increases the number of rodent liver carcinogens that are successfully predicted (p < 0.05), but also results in the prediction of increased numbers of noncarcinogenic chemicals as carcinogens. The use of multiple prechronic study endpoints provides supplementary information that enhances the predictivity of identifying chemicals with carcinogenic potential.

Oxidative Damage Precedes Nitrative Damage in Adriamycin-Induced Cardiac Mitochondrial Injury

Abstract: The purpose of the present study was to determine if elevated reactive oxygen (ROS)/nitrogen species (RNS) reported to be present in adriamycin (ADR)-induced cardiotoxicity actually resulted in cardiomyocyte oxidative/nitrative damage, and to quantitatively determine the time course and subcellular localization of these postulated damage products using an in vivo approach. B6C3 mice were treated with a single dose of 20 mg/kg ADR. Ultrastructural damage and levels of 4-hydroxy-2-nonenal (4HNE)-protein adducts and 3-nitrotyrosine (3NT) were analyzed. Quantitative ultrastructural damage using computerized image techniques showed cardiomyocyte injury as early as 3 hours, with mitochondria being the most extensively and progressively injured subcellular organelle. Analysis of 4HNE protein adducts by immunogold electron microscopy showed appearance of 4HNE protein adducts in mitochondria as early as 3 hours, with a peak at 6 hours and subsequent decline at 24 hours. 3NT levels were significantly increased in all subcellular compartments at 6 hours and subsequently declined at 24 hours. Our data showed ADR induced 4HNE-protein adducts in mitochondria at the same time point as when mitochondrial injury initially appeared. These results document for the first time in vivo that mitochondrial oxidative damage precedes nitrative damage. The progressive nature of mitochondrial injury suggests that mitochondria, not other subcellular organelles, are the major site of intracellular injury.

Emphysema and metalloelastase expression in mouse lung induced by cigarette smoke.

Abstract: Cigarette smoke (CS) causes pulmonary emphysema in humans and elastin degradation plays a key role in its pathogenesis. Previous studies on CS-exposed animals have been equivocal and have not clearly demonstrated the progression of the disease. In this study, morphometry was used to assess lung modifications to alveolar septa, airspaces, elastic and collagen fibers, and alveolar macrophages. Male (n = 40) C57/BL6 mice were exposed 3 times/day, whole body, to CS from three cigarettes for 10, 20, 30, or 60 days. Control groups (n = 10) were sham-smoked or received no exposure (day 0, n = 10). Morphometry included measurements of volume fraction of alveolar septa and airspaces, elastic and collagen fibers, and surface fraction of elastic fibers and alveolar septa. Morphometrical differences in mice after 60 days of exposure were greater than those after 10, 20, or 30 days, suggesting a progression of the disease. Inflammatory lesions in the lungs of mice contained significantly more metalloelastase (MMP-12) in macrophages at 10, 20, and 30 days than in controls of mice exposed for 60 days. These results suggest that elastin degradation took place during development of pulmonary changes in mice exposed to CS, and activation of MMPs specific for elastin may be a determining factor for susceptibility to emphysema.

Boron Supplementation Inhibits the Growth and Local Expression of IGF-1 in Human Prostate Adenocarcinoma (LNCaP) Tumors in Nude Mice

Abstract: Prostate-specific antigen (PSA) is a serine protease and one of the most abundant proteins secreted by the human prostate epithelium. PSA is used as a well-established marker of prostate cancer. The involvement of PSA in several early events leading to the development of malignant prostate tumors has made it a target for prevention and intervention. It is thought that PSA cleaves insulin-like growth factor binding protein-3 (IGFBP-3), providing increased local levels of IGF-1, leading to tumor growth. Separately, there are data that suggest an enzymatic regulatory role for dietary boron, which is a serine protease inhibitor. In this study we have addressed the use of boric acid as a PSA inhibitor in an animal study. We have previously reported that low concentrations (6 ug/mL) of boric acid can partially inhibit the proteolytic activity of purified PSA towards a synthetic fluorogenic substrate. Also, by Western blot we have followed the degradation of fibronectin by enzymatically active PSA and have found significant inhibition in the presence of boric acid. We proposed that dietary supplementation with boric acid would inhibit PSA and reduce the development and proliferation of prostate carcinomas in an animal model. We tested this hypothesis using nude mice implanted subcutaneously with LNCaP cells in Matrigel. Two groups (10 animals/group) were dosed with boric acid solutions (1.7, 9.0 mgB/kg/day) by gavage. Control group received only water. Tumor sizes were measured weekly for 8 weeks. Serum PSA and IGF-1 levels were determined at terminal sacrifice. The size of tumors was decreased in mice exposed to the low and high dose of boric acid by 38% and 25%, respectively. Serum PSA levels decreased by 88.6% and 86.4%, respectively, as compared to the control group. There were morphological differences between the tumors in control and boron-dosed animals, including a significantly lower incidence of mitotic figures in the boron-supplemented groups. Circulating IGF-1 levels were not different among groups, though expression of IGF-1 in the tumors was markedly reduced by boron treatment, which we have shown by immunohistochemistry. These data indicate that low-level dietary boron supplementation reduced tumor size and content of a tumor trophic factor, IGF-1. This promising model is being evaluated in further studies.

Trials, Tribulations, and Trends in Tumor Modeling in Mice:

Abstract: Selection of mouse models of cancer is often based simply on availability of a mouse strain and a known compatible tumor. Frequently this results in use of tumor models long on history but short on homology and quality control. Other factors including genetics, sex, immunological status, method and site of tumor implantation, technical competence, biological activity of the tumor, protocol sequence and timing, and selection of endpoints interact to produce outcomes in tumor models. Common reliance on survival and tumor burden data in a single mouse model often skews expectations towards high remission and cure rates; a finding seldom duplicated in clinical trials. Inherent limitations of tumor models coupled with the advent of new therapeutic targets reinforce need for careful attention to design, conduct, and stringent selection of in vivo and ex vivo endpoints. Preclinical efficacy testing for anti-tumor therapies should progress through a series of models of increasing sophistication that includes incorporation of genetically engineered animals, and orthotopic and combination therapy models. Pharmacology and safety testing in tumor-bearing animals may also help to improve predictive value of these models for clinical efficacy. Trends in bioinformatics, genetic refinements, and specialized imaging techniques are helping to maintain mice as the most scientifically and economically powerful model of malignant neoplasms.

Toxicoproteomics: serum proteomic pattern diagnostics for early detection of drug induced cardiac toxicities and cardioprotection.

Abstract: Proteomics is more than just generating lists of proteins that increase or decrease in expression as a cause or consequence of pathology. The goal should be to characterize the information flow through the intercellular protein circuitry which communicates with the extracellular microenvironment and then ultimately to the serum/plasma macroenvironment. The nature of this information can be a cause, or a consequence, of disease and toxicity based processes as cascades of reinforcing information percolate through the system and become reflected in changing proteomic information content of the circulation. Serum Proteomic Pattern Diagnostics is a new type of proteomic platform in which patterns of proteomic signatures from high dimensional mass spectrometry data are used as a diagnostic classifier. While this approach has shown tremendous promise in early detection of cancers, detection of drug-induced toxicity may also be possible with this same technology. Analysis of serum from rat models of anthracycline and anthracenedione induced cardiotoxicity indicate the potential clinical utility of diagnostic proteomic patterns where low molecular weight peptides and protein fragments may have higher accuracy than traditional biomarkers of cardiotoxicity such as troponins. These fragments may one day be harvested by circulating nanoparticles designed to absorb, enrich and amplify the diagnostic biomarker repertoire generated even at the critical initial stages of toxicity.

Ferruginous bodies: implications in the mechanism of fiber and particle toxicity.

Abstract: Exposures to fibers and particles can be associated with several different lung injuries including bronchitis, bronchiolitis, pneumonitis, pleuritis, pulmonary alveolar proteinosis, pneumoconiosis, mesotheliomas, and lung cancers. The mechanism of biological effect exerted by fibers and particles has not been exactly defined. Exposures to all fibers and particles introduce a solid-liquid interface into the lower respiratory tract. These surfaces all have some concentration of oxygen-containing functional groups that demonstrate a capacity to coordinate iron. Radical generation is catalyzed by this metal resulting in a cascade of cell signaling, transcription factor activation, and mediator release. We propose that the ferruginous body (i.e., a fiber or particle with a coating of both protein and iron) provides direct evidence of a participation of iron in the biological effect of both fibers and particles. It is recommended that an identification of ferruginous bodies in the lung be regarded as support for a metal-catalyzed oxidative stress in the mechanism of cell and tissue injury.

Early occurrence of spontaneous tumors in CD-1 mice and Sprague-Dawley rats.

Abstract: It is sometimes difficult to assess the relevance of tumors that occur in treated animals in short-term studies. This report is intended to establish a general profile of tumor occurrence in young control CD-1 mice and Sprague-Dawley rats. Data from 20 rat and 20 mouse carcinogenicity studies conducted between 1990 and 2002 at Huntingdon Life Sciences, UK. were collected and evaluated. The route of administration was either dietary oral gavage, and the analysis was confined to sporadic deaths (decedents) in control groups occurring during the first 50 weeks of study. In addition, tumor occurrence between 50-80 weeks were compared. In mice, the most common tumor was lymphoma, followed by bronchiolo-alveolar adenoma. In rats, the most common tumor was adenoma of the pituitary gland, followed by mammary fibroadenoma, and adenocarcinoma. When studies of up to 50 weeks, between 50 and 80 weeks, and at 2-year termination were compared, there was no great difference in tumor occurrence except in male rats, in which the most common tumor up to 50 weeks on study was lymphoma, whereas the most common tumor between 50-80 weeks and at 2 years was pituitary adenoma.

The toxicity of SCH 351591, a novel phosphodiesterase-4 inhibitor, in Cynomolgus monkeys.

Abstract: SCH351591, a novel phosphodiesterase-4 inhibitor under investigation as a potential therapeutic for asthma and chronic obstructive pulmonary disease (COPD), was evaluated in a 3-month rising-dose study in Cynomolgus monkeys. Four groups, containing four monkeys/sex, received vehicle control or rising doses up to 12, 24, or 48 mg/kg of SCH351591 daily. Although initial exposure produced clinical signs of emesis, reduced food intake, and reduced body weight, tachyphylaxis to the emesis allowed dose escalation up to 48 mg/kg/day. Two monkeys died and 3 were sacrificed in moribund condition over the course of the study. Early mortality, involving monkeys dosed with 12 or 24 mg/kg, was attributed to sepsis (2 monkeys) or colon inflammation (3 monkeys). Leukocyte function assays on low- and mid-dose group survivors revealed an inhibition of T lymphocyte proliferation for 12 mg/kg group males and 24 mg/kg group monkeys of both sexes. Necropsy findings, unassociated with early mortality, included reduced size and weight of the thymus, depletion of body fat, red discoloration of the gastric mucosa, and perivascular hemorrhage of the stomach and heart. Stomach and heart gross findings were present in the high-dose group only. Histopathologic lesions, in addition to those attributed to concurrent bacterial infection, included thymic atrophy, serous atrophy of fat, myocardial degeneration and acute to chronic inflammation of small to medium-sized arteries in various organs and tissues including the heart, kidneys, stomach, salivary glands, pancreas, esophagus, gallbladder, and mesentery. The findings of this study demonstrate the potential of a PDE4 inhibitor to alter immunologic response as well as to produce arteriopathy in nonhuman primates.

Application of Toxicogenomics to Toxicology: Basic Concepts in the Analysis of Microarray Data

Abstract: Toxicology and the practice of pathology are rapidly evolving in the postgenomic era. Observable treatment related changes have been the hallmark of toxicology studies. Toxicogenomics is a powerful new tool that may show gene and protein changes earlier and at treatment levels below the limits of detection of traditional measures of toxicity. It may also aid in the understanding of toxic mechanisms. It is important to remember that it is only a tool and will provide meaningful results only when properly applied. As is often the case with new experimental tools, the initial utilization is driven more by the technology than application to problem solving. Toxicogenomics is interdisciplinary in nature including at a minimum, pathology, toxicology, and genomics. Most studies will require the input from the disciplines of toxicology, pathology, molecular biology, bioinformatics, biochemistry, and others depending on the types of questions being asked.

Relevance of Animal Carcinogenesis Findings to Human Cancer Predictions and Prevention

Abstract: Use of laboratory animals to identify carcinogenic potential of chemicals, mixtures, and other agents has a modern history of greater than 40 years from which much useful scientific and public health information can be derived. While laboratory animals differ from humans in some respects that may affect responses to hazardous exposures, use of such models is based on experimental evidence indicating that there are more genetic, genomic, physiological, biochemical, and metabolic similarities than differences among mammalian species. Issues of concordance of responses between rodent species and between rodents and humans as well as repeatability and site-specificity are important considerations in evaluating laboratory animal carcinogenicity results. Variables in experimental design such as animal strain, diet, route of exposure, and study duration as well as single-site versus multisite carcinogenic responses all influence interpretation and intelligent use of study data. Similarities and differences in site-specific laboratory animal and corresponding human cancers should also be considered in study evaluation. Recent attempts to explore genetically engineered mice and to humanize the mouse for more relevant identification of carcinogen hazard identification have yielded mixed results. In the end we are confronted by the realization that virtually all animal cancer models are useful but imperfect surrogates for humans. Assuming the percentage of chemicals currently in commerce that are estimated to be potent animal or human carcinogens is quite low, the task of identifying agents with significant carcinogenic potential is daunting and important. The biological conundrum of scientific debate regarding the relevance of carcinogenicity studies in laboratory animals is likely to continue. Nonetheless public health considerations must take precedence when deciding human safety issues.

Effects of dietary isoflavone aglycones on the reproductive tract of male and female mice.

Abstract: We assessed the effects of dietary consumption of soy isoflavone aglycones on the reproductive tract of sexually mature male and female mice. Isoflavone concentrates with a ratio of 10:1, 2:1 or 1:10 genistein:daidzein (G:D) were added to provide 120 mg total isoflavones/1800 Calories (approximately 40 mg/kg body weight) to diets having either casein/lactalbumin or soy protein isolate as the source of protein. After 16 weeks, mice were necropsied and gross and histopathologic assessments of uterus, vagina, testes and accessory sex glands were completed. Effects of the 10G:1D isoflavone concentrates were absent or minimal in females but in males included atrophy of accessory sex glands. In contrast, the 2G:1D and 1G:10D concentrates caused dramatic estrogenic effects in both male and female mice. Effects in females included endometritis and effects typical of estrogenic stimulation (i.e., uterine enlargement, keratinization of vaginal epithelium, increased height of endometrial surface epithelial cells, and uterine squamous metaplasia). Effects in males included reduced plasma testosterone concentrations, atrophy of seminiferous epithelium, atrophy of accessory sex glands, and squamous metaplasia of seminal vesicles. Some effects varied with protein source. We conclude that a diet containing approximately 40 mg/kg soy isoflavone aglycones with a genistein:daidzein ratio of 2:1 or less has marked estrogenic effects on the reproductive system of male and female mice.

Development and use of biomarkers in oncology drug development.

Abstract: Successful development and use of biomarkers will improve the productivity of oncology drug development. Recognition of the importance of biomarkers for speeding drug development is reflected in the precise definitions and concepts proposed by an NIH Working Group to standardize terminology and promote a more coherent and systematic approach to the development and use of biomarkers. Potential clinical biomarkers of drug efficacy are often identified through pre-clinical studies or basic research. Identification of potential biomarkers for use in oncology is moving rapidly forward through continuing advances in clinical imaging technologies, especially molecular and functional imaging. Other rapid advances are a product of the growing availability of new scientific reagents for established technologies and of high-throughput genomic and proteomic technologies that can generate hundreds of potential biomarkers for further evaluation. In certain cases, conventional clinical diagnostic techniques or assays can be adapted for use in pre-clinical models to evaluate their ability to serve as biomarkers for predicting clinical responses to new drug candidates. Evaluation (pre-clinical and clinical) of a potential biomarker is often the longest stage of biomarker development, and standards for evaluation or validation depend on the intended use and stage of clinical development. Biomarkers verified for use in preclinical studies can be used to help select appropriate animal models and lead compounds. Biomarkers verified for use in clinical trials can confirm a drug's pharmacological or biological mechanism of action, guide protocol design, aid patient and dose selection, and help to minimize safety risks. Oncology drug development can be optimized by using a tiered set of clinical biomarkers that predict compound efficacy and safety with increasing confidence at each rise in tier thereby aiding corporate decision-making about advancing compounds. In oncology, a special class of extensively evaluated biomarkers of efficacy (surrogate endpoints) that generally correlate with desired clinical outcomes can be used as a basis for corporate decisions as well as for gaining accelerated provisional regulatory approval of a drug.

Use of transgenic mice in carcinogenicity hazard assessment.

Abstract: Determining the carcinogenic potential of materials to which humans have significant exposure is an important, complex and imperfect exercise. Not only are the methods for such determinations protracted, expensive and utilize large numbers of animals, extrapolation of data from such studies to human risk is imprecise. Toxicologists have long recognized these shortcomings but the 2-year chronic rodent study has remained the gold standard. Recent developments in the field of molecular oncology and development of methods to insert or inactivate specific genes in animals have provided the tools with which to develop the next generation of carcinogenicity assays. With improved understanding of oncogene activation and tumor suppressor gene inactivation a number of animal models have been developed to dramatically reduce latency for chemically induced cancers. This has led to the development of shorter carcinogenicity assays. Also, because the spontaneous tumor frequencies in these animals are low during the in-life portion of the study, and studies are terminated well before the health complications of advanced aging are observed, it has been possible to reduce the group sizes and reduce animal usage. FDA's adoption of ICH S1B in 1997, (ICH, 1997) "Testing for the Carcinogenicity of Pharmaceuticals," opened the door for the use of such transgenic models in regulatory toxicology. This presentation reviews the current state of the science and its application to regulatory issues.

Exploration of low-dose estrogen effects: identification of No Observed Transcriptional Effect Level (NOTEL).

Abstract: Identifying a minimal dose capable of eliciting a biological response is a fundamental issue in a number of scientific fields, including: drug development, signal transduction research, and environmental toxicology. Frequently, proliferation, viability, and other assays based on the cellular response to a treatment are used to assess the threshold dose for minimal activity. Here we propose a novel approach for identifying the effects of low dose treatments and pinpointing the threshold dose. Using microarrays, we examined the transcriptional response of a hormone responsive breast cancer cell line (MCF-7) stimulated with various concentrations of estrogen. Previous studies have focused on transcriptional responses to physiologically relevant concentrations of estrogen. However, relatively few studies have examined the transcriptional effects of concentrations below normal physiologic levels. These doses may not stimulate the expression of any genes or, alternatively, may regulate a different subset of genes that had not been previously characterized as estrogen responsive. We used gene expression profiling, coupled with a detailed analysis of replicates, to measure estrogen effects on many transcriptional targets and found that only physiologically relevant doses of estrogen (1 × 10 - 1 0 M and higher) were capable of inducing a transcriptional response. This study demonstrates the utility of gene expression profiling as a means to identify concentrations that do not elicit a change in gene expression, or simply a No Observed Transcriptional Effect Level (NOTEL). The identification of a NOTEL for a given compound may be beneficial in several different scientific disciplines. For example, in the development of therapeutic drugs, a NOTEL could be used to identify doses of pharmaceutical compounds that are no longer effective at modulating the expression of biomarkers of efficacy.

Decreased Longevity and Enhancement of Age-Dependent Lesions in Mice Lacking the Nuclear Receptor Peroxisome Proliferator-Activated Receptor α (PPARα)

Abstract: The nuclear receptor peroxisome proliferator-activated receptor alpha (PPARalpha) is activated by peroxisome proliferators (PP), a large class of structurally diverse xenobiotic chemicals, hypolipidemic drugs, and endogenous lipids. PPARalpha alters the transcriptional programs of genes whose functions include lipid metabolism, inflammation, cell fate, and stress responses in liver, heart, kidney, and skin. Many of these genes are also under control of PPARalpha in the absence of exogenous peroxisome proliferator exposure. Mice that lack PPARalpha (PPARalpha-null mice) exhibit a number of defects in lipid metabolism and accumulate lipids in the liver. Here, we compared the age-dependent lesions in the liver, kidney, and heart in PPARalpha-null mice with those observed in wild-type SV129 mice, in the absence of exogenous chemical exposure. Groups of mice were sacrificed, at 6, 12, 18, 21, or 24 months of age, or allowed to age until moribund or found dead. PPARalpha-null mice had decreased longevity, due to a variety of causes. Statistically significant differences in the occurrence of a number of lesions between strains was observed. Hepatocellular carcinomas and multiple hepatocellular adenomas occurred in PPARa-null mice but not wild type mice. Various nonneoplastic spontaneous aging lesions occurred at higher incidence, shorter latency, or increased severity in PPARalpha-null mice compared with wild-type mice. In the liver, these included vacuolated hepatocytes and sinusoidal cells and mixed cell inflammation. The kidneys of PPARalpha-null mice exhibited higher incidences and severities of cortical mineralization. Minimal myocardial mineralization occurred at a higher incidence in PPARalpha-null mice. Our results imply that PPARalpha delays the development of some spontaneous lesions associated with aging in the liver, kidney, and heart of SV129 mice.

Carcinogenic Effect of N-Nitrosodimethylamine on Diploid and Triploid Zebrafish (Danio rerio)

Abstract: Viability of polyploid organisms in lower vertebrates including fish provides an additional tool to investigate genetic mechanisms of neoplastic transformation caused by carcinogens. Here we present data on differential sensitivity of diploid and triploid zebrafish (Danio rerio) to N-nitrosodimethylamine (NDMA) induced hepatocarcinogenesis. The effect of the carcinogen was studied in 100 diploid and 120 triploid zebrafish. Zebrafish, age 5-6 weeks, were exposed to 50 ppm NDMA for 8 weeks and then were transferred into fresh carcinogen-free water until necropsy. At the necropsy performed 24 weeks after beginning the treatment, cholangiolar tumors (cholangiocarcinomas and cholangiomas) were essentially observed in diploid zebrafish only, while the incidence of hepatocellular tumors (hepatocellular carcinomas and adenomas) was similar in diploid and triploid zebrafish, 7.7% and 9.5%, respectively. By contrast, 36 weeks after beginning the treatment, the incidence of hepatocellular tumors was significantly lower in diploid animals as compared to triploid ones, 10.3% and 33.8%, respectively. The incidence of cholangiolar tumors in diploid and triploid zebrafish was not significantly different, 10.3% and 14.9%, respectively. Therefore, the increase of ploidy appeared to have a differential effect on the induction of these 2 types of liver tumors in zebrafish. This finding suggests a difference in genetic mechanisms of the tumor development revealed by utilization of triploid animals in this study. However, triploid zebrafish demonstrated an overall increase in latency period in the development of both types of hepatic tumors, a finding that can be interpreted as an increased resistance of triploid animals to the carcinogenic effect of NDMA.

Protective activity of hesperidin and lipoic acid against sodium arsenite acute toxicity in mice.

Abstract: The objective of the present work was to evaluate the toxic effects of sodium arsenite, As(III), in mice and the protective effect of 2 antioxidants, hesperidin and lipoic acid, against the observed As(III)-induced toxicity. In each study, mice were assigned to 1 of 4 groups: control, antioxidant, antioxidant + arsenite, and arsenite. Animals were first injected with the vehicle or 25 mg antioxidant/kg BW. After 30 minutes they received an injection of 10 mg arsenite/kg BW or 0.9% NaCl. Two hours after the first injection, the liver, kidney, and testis were collected for histological evaluation. Liver samples were also taken for quantification of arsenic. In mice exposed only to As(III), various histopathological effects were observed in the liver, kidneys, and testes. In mice pretreated with either hesperidin or lipoic acid, a reduction of histopathologic effects on the liver and kidneys was observed. No protective effects were observed in the testes for either of the 2 studied antioxidants. In conclusion, hesperidin and lipoic acid provided protective effects against As(III)-induced acute toxicity in the liver and kidneys of mice. These compounds may potentially play an important role in the protection of populations chronically exposed to arsenic.

Antibodies that label paraffin-embedded mouse tissues: a collaborative endeavor.

Abstract: Histology and immunohistochemistry are important tools in the study of human diseases and their respective animal models. The study of mouse models has been hampered by the absence of a large set of mouse-specific antibodies adapted to paraffin-embedded tissues. A total of 196 antibodies were tested on paraffin-embedded mouse tissues preserved in five different fixatives (Fekete's acid-alcohol-formalin, 10% neutral buffered formalin, 4% paraformaldehyde, IHC Zinc Fixative, and Bouin's fixative). The antibodies were targeted to proteins of the cytoplasm (n = 100), plasma membrane (n = 48), nucleus (n = 36), extracellular compartment (n = 5), cytoplasm/cell membrane (n = 4), and viral proteins (n = 3). A total of 83 antibodies provided an adequate signal to noise ratio. Of these, adequate labeling required heat-mediated epitope retrieval or enzymatic digestion for 32 and 8 antibodies, respectively. Epitope recognition was best for tissues fixed with Fekete's acid-alcohol-formalin. However, some proteins could be detected only in IHC Zinc Fixative, confirming that there is no single fixative suitable for the preservation of all epitopes. Four of 13 antibodies that failed to label their cellular targets on tissue sections successfully labeled whole-mount tissues, indicating that tissue processing plays an important role in epitope degradation. Regularly updated information on immunohistochemistry of normal and neoplastic mouse tissues is accessible online at (http://tumor.informatics.jax.org); links to antibody suppliers' web sites are provided.

Immunohistochemical Detection of Activated Caspases in Apoptotic Hepatocytes in Rat Liver

Abstract: In our study we tested the utility of antibodies that specifically recognize the cleaved large (active) subunits of caspase-3 and caspase-9 for immunohistochemical detection of apoptotic hepatocytes in rat liver sections using archival material from cyproterone acetate (CPA)-treated and control rats. CPA blocks apoptosis of hepatocytes and discontinuation of CPA treatment results in a syncronized wave of hepatocyte apoptosis. By comparing liver sections from CPA-treated and control rats with high and low rates of apoptosis we observed a close correlation between the occurrence of cleaved caspase-positive apoptotic figures and H&E-stained apoptotic bodies when evaluated in parallel sections. Caspase-stained figures were either immuno-positive apoptotic bodies or pre-apoptotic hepatocytes showing cytoplasmic and/or nuclear caspase-staining with otherwise normal cellular appearance. In extension of these observations we developed a double-immunohistochemical staining procedure which enables the detection of caspase-3-positive apoptotic hepatocytes within glutathione-S-transferase-P (GST-P)-positive preneoplastic liver foci. By use of this technique, inhibition of apoptosis by 2,3,7,8-tetrachlorodibenzo- p-dioxin as detected by counting of H&E-stained apoptotic bodies was found to correlate with a strong reduction of cleaved caspase-positive hepatocytes in GST-P-positive preneoplastic foci. In summary, this study demonstrates that cleaved caspase-positive apoptotic hepatocytes could be reliably identified and quantified both in normal and neoplastically transformed liver tissue.