Robert R. Maronpot

Effects of fixation on RNA extraction and amplification from laser capture microdissected tissue.

One of the key end points for understanding the molecular basis of carcinogenesis is the quantitation of gene expression in specific cell populations. Microdissection techniques allow extraction of morphologically distinct cells for molecular analysis. A recent advance in microdissection uses the PixCell laser capture microdissection (LCM) system, which allows for precise removal of pure cell populations from morphologically preserved tissue sections. The objective of this study was to determine the optimal fixation protocol for analyzing RNA from tissue samples using LCM. Optimal fixation must provide acceptable morphology, allow proper laser capture of selected cells, and preserve the integrity of mRNA. We evaluated the effects of both cross‐linking and precipitive‐type fixatives on frozen and paraffin‐embedded mouse liver tissue. For assessment of the quality of the mRNA in LCM samples generated from various fixed tissues, reverse transcription–polymerase chain reaction (RT‐PCR)–amplified mouse liver β2‐microglobulin mRNA was detected with ethidium bromide. We also examined mouse glyceraldehyde‐3‐phosphate‐dehydrogenase by using the fluorogenic TaqMan system for real‐time quantitative detection of RT‐PCR products. Frozen tissues yielded more RT‐PCR product than did paraffin‐embedded tissues. In both frozen and paraffin‐embedded tissues, differences were observed between the fixatives. Precipitive fixatives, such as ethanol and acetone, consistently produced more RT‐PCR amplification product than did cross‐linking fixatives such as formalin. Optimal fixation protocols for LCM analysis will facilitate the examination of gene expression in specific cell populations, accelerating investigations of the molecular differences responsible for the phenotypic changes observed during carcinogenesis. Mol. Carcinog. 25:86–91, 1999. Published 1999 Wiley‐Liss, Inc.

The Orphan Nuclear Receptor Constitutive Active/Androstane Receptor Is Essential for Liver Tumor Promotion by Phenobarbital in Mice

Hepatocellular carcinoma (HCC) is known to progress through a step often called tumor promotion. Phenobarbital (PB) is the prototype of nongenotoxic cacinogens that promote HCC in rodents. The molecular target of PB to elicit the promotion has been the subject of intense investigations over the last 30 years since it was discovered. The nuclear receptor constitutive active/androstane receptor (CAR) is activated by PB as well as by various other xenobiotics such as therapeutic drugs and environmental pollutants. CAR activation results in the transcriptional induction of numerous hepatic genes including those that encode xenobiotic-metabolizing enzymes such as a set of cytochrome P450s. In addition to PB, many CAR activators are nongenotoxic carcinogens, but the role of CAR in liver tumor promotion remains unexplored. Using Car−/− mice, we have here examined tumor promotion by chronic treatment with PB in drinking water after tumor initiation with a single dose of the genotoxic carcinogen diethylnitrosamine. None of the Car−/− mice developed either eosinophilic foci or advanced liver tumors, whereas all Car+/+ mice developed HCC and/or adenoma by 39 weeks. The results indicate that CAR is the molecular target of promotion by PB and that activation of this receptor is an essential requirement for liver tumor development.

Brain Inflammation and Alzheimer's-Like Pathology in Individuals Exposed to Severe Air Pollution

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 Alzheimers-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 β-amyloid (Aβ42), 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-κB 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 Aβ42 compared to residents in low air pollution cities. Increased COX2 expression and Aβ42 accumulation were also observed in the olfactory bulb. These findings suggest that exposure to severe airpollution is associated with brain inflammation and Aβ 42 accumulation, two causes of neuronal dysfunction that precede the appearance of neuritic plaques and neurofibrillary tangles, hallmarks of Alzheimers disease.

Classification of Proliferative Pulmonary Lesions of the Mouse Recommendations of the Mouse Models of Human Cancers Consortium

Rapid advances in generating new mouse genetic models for lung neoplasia provide a continuous challenge for pathologists and investigators. Frequently, phenotypes of new models either have no precedents or are arbitrarily attributed according to incongruent human and mouse classifications. Thus, comparative characterization and validation of novel models can be difficult. To address these issues, a series of discussions was initiated by a panel of human, veterinary, and experimental pathologists during the Mouse Models of Human Cancers Consortium (NIH/National Cancer Institute) workshop on mouse models of lung cancer held in Boston on June 20–22, 2001. The panel performed a comparative evaluation of 78 cases of mouse and human lung proliferative lesions, and recommended development of a new practical classification scheme that would (a) allow easier comparison between human and mouse lung neoplasms, (b) accommodate newly emerging mouse neoplasms, and (c) address the interpretation of benign and preinvasive lesions of the mouse lung. Subsequent discussions with additional experts in pulmonary pathology resulted in the current proposal of a new classification. It is anticipated that this classification, as well as the complementary digital atlas of virtual histological slides, will help investigators and pathologists in their characterization of new mouse models, as well as stimulate further research aimed at a better understanding of proliferative lesions of the lung.

Proliferative and Nonproliferative Lesions of the Rat and Mouse Hepatobiliary System

The INHAND Project (International Harmonization of Nomenclature and Diagnostic Criteria for Lesions in Rats and Mice) is a joint initiative of the Societies of Toxicologic Pathology from Europe (ESTP), Great Britain (BSTP), Japan (JSTP) and North America (STP) to develop an internationally-accepted nomenclature for proliferative and non-proliferative lesions in laboratory animals. The purpose of this publication is to provide a standardized nomenclature and differential diagnosis for classifying microscopic lesions observed in the hepatobiliary system of laboratory rats and mice, with color microphotographs illustrating examples of some lesions. The standardized nomenclature presented in this document is also available for society members electronically on the internet (http://goreni.org). Sources of material included histopathology databases from government, academia, and industrial laboratories throughout the world. Content includes spontaneous and aging lesions as well as lesions induced by exposure to test materials. A widely accepted and utilized international harmonization of nomenclature for lesions of the hepatobiliary system in laboratory animals will decrease confusion among regulatory and scientific research organizations in different countries and provide a common language to increase and enrich international exchanges of information among toxicologists and pathologists.

Air Pollution, Cognitive Deficits and Brain Abnormalities: A Pilot Study with Children and Dogs.

Exposure to air pollution is associated with neuroinflammation in healthy children and dogs in Mexico City. Comparative studies were carried out in healthy children and young dogs similarly exposed to ambient pollution in Mexico City. Children from Mexico City (n: 55) and a low polluted city (n:18) underwent psychometric testing and brain magnetic resonance imaging MRI. Seven healthy young dogs with similar exposure to Mexico City air pollution had brain MRI, measurement of mRNA abundance of two inflammatory genes cyclooxygenase-2, and interleukin 1 beta in target brain areas, and histopathological evaluation of brain tissue. Children with no known risk factors for neurological or cognitive disorders residing in a polluted urban environment exhibited significant deficits in a combination of fluid and crystallized cognition tasks. Fifty-six percent of Mexico City children tested showed prefrontal white matter hyperintense lesions and similar lesions were observed in dogs (57%). Exposed dogs had frontal lesions with vascular subcortical pathology associated with neuroinflammation, enlarged Virchow-Robin spaces, gliosis, and ultrafine particulate matter deposition. Based on the MRI findings, the prefrontal cortex was a target anatomical region in Mexico City children and its damage could have contributed to their cognitive dysfunction. The present work presents a groundbreaking, interdisciplinary methodology for addressing relationships between environmental pollution, structural brain alterations by MRI, and cognitive deficits/delays in healthy children.

Best Practices Guideline: Toxicologic Histopathology:

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

An enhancement method for immunohistochemical staining of proliferating cell nuclear antigen in archival rodent tissues

An enhanced immunohistochemical procedure to detect proliferating cell nuclear antigen (PCNA), an endogenous cell replication marker, has been successfully applied to formalin-fixed, paraffin-embedded archival rat and mouse tissues. The procedure involves microwave oven heating of tissue sections in a commercially available antigen retrieval solution of heavy metal salts. Successful immunohistochemical staining of PCNA can be consistently obtained in tissues fixed for over 24 months in formalin and in sections made from paraffin blocks stored in our tissue archives for up to 19 months. Use of this technique will allow retrospective staining of rodent tissues for identification of S phase cells as an indication of DNA replicative activity in previously conducted toxicity and carcinogenicity studies.

Spontaneous Lesions in Aging FVB/N Mice

The FVB/N mouse strain was created in the early 1970s and has since been used extensively in transgenic research because of its well-defined inbred background, superior reproductive performance, and prominent pronuclei of fertilized zygotes, which facilitate microinjection of DNA. Little is known, however, about the survivability and spontaneous disease of nontransgenic FVB/N mice. Therefore, the purpose of this study was to determine survival to 24 mo of age and the incidence of neoplastic and nonneoplastic disease at 14 and 24 mo of age. At 14 mo of age, the incidence of tumor-bearing mice was 13% in males (n = 45) and 26% in females (n = 98). All tumors in males and most in females at this time were alveolar-bronchiolar (AB) neoplasms of the lung. Survival to 24 mo of age was approximately 60% in both sexes (29/50 males, 71/116 females), and the incidence of mice with tumors at this time was 55% in males and 66% in females. In decreasing order of frequency, the following neoplasms were observed in >5% of subjects: in males, lung AB tumors, liver hepatocellular tumors, subcutis neural crest tumors, and Harderian gland adenomas; in females, lung AB tumors, pituitary gland adenomas, ovarian tumors (combined types), lymphomas, histiocytic sarcomas, Harderian gland adenomas, and pheochromocytomas. Compared with other mouse strains, the observed incidences of tumors in FVB/N mice suggest a higher than usual rate of lung tumors and a lower than usual incidence of liver tumors and lymphomas. This tumor profile should be considered in the interpretation of neoplastic phenotypes in FVB/N-derived transgenic lines.

DNA Damage in Nasal and Brain Tissues of Canines Exposed to Air Pollutants Is Associated with Evidence of Chronic Brain Inflammation and Neurodegeneration

Acute, subchronic, or chronic exposures to particulate matter (PM) and pollutant gases affect people in urban areas and those exposed to fires, disasters, and wars. Respiratory tract inflammation, production of mediators of inflammation capable of reaching the brain, systemic circulation of PM, and disruption of the nasal respiratory and olfactory barriers are likely in these populations. DNA damage is crucial in aging and in age-associated diseases such as Alzheimers disease. We evaluated apurinic/apyrimidinic (AP) sites in nasal and brain genomic DNA, and explored by immunohistochemistry the expression of nuclear factor NFκB p65, inducible nitric oxide synthase (iNOS), cyclo-oxygenase 2 (COX2), metallothionein I and II, apolipoprotein E, amyloid precursor protein (APP), and beta-amyloid1-42 in healthy dogs naturally exposed to urban pollution in Mexico City. Nickel (Ni) and vanadium (V) were measured by inductively coupled plasma mass spectrometry (ICP-MS). Forty mongrel dogs, ages 7 days—10 years were studied (14 controls from Tlaxcala and 26 exposed to urban pollution in South West Metropolitan Mexico City (SWMMC)). Nasal respiratory and olfactory epithelium were found to be early pollutant targets. Olfactory bulb and hippocampal AP sites were significantly higher in exposed than in control age matched animals. Ni and V were present in a gradient from olfactory mucosa > olfactory bulb > frontal cortex. Exposed dogs had (a) nuclear neuronal NFκB p65, (b) endothelial, glial and neuronal iNOS, (c) endothelial and glial COX2, (d) ApoE in neuronal, glial and vascular cells, and (e) APP and β amyloid1-42 in neurons, diffuse plaques (the earliest at age 11 months), and in subarachnoid blood vessels. Increased AP sites and the inflammatory and stress protein brain responses were early and significant in dogs exposed to urban pollution. Oil combustion PM-associated metals Ni and V were detected in the brain. There was an acceleration of Alzheimers-type pathology in dogs chronically exposed to air pollutants. Respiratory tract inflammation and deteriorating olfactory and respiratory barriers may play a role in the observed neuropathology. These data suggest that Alzheimers disease may be the sequela of air pollutant exposures and the resulting systemic inflammation.