Frequent aberrant methylation of p16INK4a in primary rat lung tumors.

The p16INK4a (p16) tumor suppressor gene is frequently inactivated by homozygous deletion or methylation of the 5' CpG island in cell lines derived from human non-small-cell lung cancers. However, the frequency of dysfunction in primary tumors appears to be significantly lower than that in cell lines. This discordance could result from the occurrence or selection of p16 dysfunction during cell culture. Alternatively, techniques commonly used to examine tumors for genetic and epigenetic alterations may not be sensitive enough to detect all dysfunctions within the heterogeneous cell population present in primary tumors. If p16 inactivation plays a central role in development of non-small-cell lung cancer, then the frequency of gene inactivation in primary tumors should parallel that observed in cell lines. The present investigation addressed this issue in primary rat lung tumors and corresponding derived cell lines. A further goal was to determine whether the aberrant p16 gene methylation seen in human tumors is a conserved event in this animal model. The rat p16 gene was cloned and sequenced, and the predicted amino acid sequence of its product found to be 62% homologous to the amino acid sequence of the human analog. Homozygous deletion accounted for loss of p16 expression in 8 of 20 cell lines, while methylation of the CpG island extending throughout exon 1 was observed in 9 of 20 cell lines. 2-Deoxy-5-azacytidine treatment of cell lines with aberrant methylation restored gene expression. The methylated phenotype seen in cell lines showed an absolute correlation with detection of methylation in primary tumors. Aberrant methylation was also detected in four of eight primary tumors in which the derived cell line contained a deletion in p16. These results substantiate the primary tumor as the origin for dysfunction of the p16 gene and implicate CpG island methylation as the major mechanism for inactivating this gene in the rat lung tumors examined. Furthermore, rat lung cancer appears to be an excellent model in which to investigate the mechanisms of de novo gene methylation and the role of p16 dysfunction in the progression of neoplasia.

Comparative carcinogenic effects of nickel subsulfide, nickel oxide, or nickel sulfate hexahydrate chronic exposures in the lung.

The relative toxicity and carcinogenicity of nickel sulfate hexahydrate (NiSO4.6H2O), nickel subsulfide (Ni3S2), and nickel oxide (NiO) were studied in F344/N rats and B6C3F1 mice after inhalation exposure for 6 h/day, 5 days/week for 2 years. Nickel subsulfide (0.15 and 1 mg/m3) and nickel oxide (1.25 and 2.5 mg/m3) caused an exposure-related increased incidence of alveolar/bronchiolar neoplasms and adrenal medulla neoplasms in male and female rats. Nickel oxide caused an equivocal exposure-related increase in alveolar/bronchiolar neoplasms in female mice. No exposure-related neoplastic responses occurred in rats or mice exposed to nickel sulfate or in mice exposed to nickel subsulfide. These findings are consistent with results from other studies, which show that nickel subsulfide and nickel oxide reach the nucleus in greater amounts than the do water-soluble nickel compounds such as nickel sulfate. It has been proposed that the more water-insoluble particles are phagocytized, whereas the vacuoles containing nickel migrate to the nuclear membrane, where they release nickel ions that effect DNA damage. The findings from these experimental studies show that chronic exposure to nickel can cause lung neoplasms in rats, and that this response is related to exposure to specific types of nickel compounds.

Absence of p53 gene mutations in rat colon carcinomas induced through the synergistic interaction between methylazoxymethanol and X-irradiation.

p53 is one the most frequently mutated genes found in human colonic tumors. Because colonic neoplasms induced in rats by certain chemical carcinogens are similar to human colonic tumors in their histological features and proliferation characteristics, the rat has been used as an experimental model to study the pathogenesis of colon cancer. However, p53 mutations were not detected in the chemically induced colonic tumors analyzed for p53 mutations. X-irradiation has also been shown to induce colonic neoplasms in rats that resemble human colonic tumors histopathologically. Because the incidence of colonic tumors induced by methylazoxymethanol (MAM) in rats was shown to be enhanced by X-irradiation, we immunohistochemically analyzed these colonic carcinomas for the presence of p53 gene mutations. The immunohistochemical analyses clearly showed the absence of nuclear immunoreactivity in all ten tumors examined. The results from the present study indicate that point mutations in p53, at least in the coding region, are not involved in the development of colon cancer induced by the combination of MAM and X-irradiation. Our observations, together with the data from previous studies, further suggest that rat colon carcinogenesis, unlike human colon cancer, may not involve p53 mutation as an obligatory event.

Failure of cigarette smoke to induce or promote lung cancer in the A/J mouse.

A six-month bioassay in A/J mice was conducted to test the hypothesis that chronically inhaled mainstream cigarette smoke would either induce lung cancer or promote lung carcinogenicity induced by the tobacco-specific nitrosamine, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). Groups of 20 female A/J mice were exposed to filtered air (FA) or cigarette smoke (CS), injected with NNK, or exposed to both CS and NNK. At 7 weeks of age, mice were injected once with NNK; 3 days later, they were exposed to CS for 6 h/day, 5 days/week, for 26 weeks at a mean 248 mg total particulate matter/m3 concentration. Animals were sacrificed 5 weeks after exposures ended for gross and histological evaluation of lung lesions. No significant differences in survival between exposure groups was observed. A biologically significant level of CS exposure was achieved as indicated by CS-induced body weight reductions, lung weight increases, and carboxyhemoglobin levels in blood of about 17%. Crude tumor incidences, as determined from gross observation of lung nodules, were similar between the CS-exposed and FA groups, and the NNK and CS + NNK groups. Incidences in either of these latter groups were greater than either the CS or FA groups. Furthermore, tumor multiplicity in tumor-bearing animals was not significantly different among any of the three groups (FA, NNK, CS + NNK) in which tumors were observed. Thus, CS exposure neither induced lung tumors nor promoted NNK-induced tumors. Because the CS exposure concentration was probably near the maximally tolerable level, longer exposures should be evaluated to potentially establish a CS-induced model of lung carcinogenesis in the A/J mouse.

Influence of exposure concentration or dose on the distribution of particulate material in rat and human lungs.

Differences among species in the anatomic sites of particle retention could influence responses to inhaled particles. In this study, we used morphometric techniques to examine the influence of exposure concentration on particle retention in histologic sections from rats and humans. The rats had been exposed for 24 months to diesel exhaust at 0.35, 3.5, or 7.0 mg soot/m(3). The human subjects were nonsmokers who did not work as miners, nonsmoking coal miners who worked under the current standard of 2 mg dust/m(3) for 10-20 years (mean = 14 years), and nonsmoking coal miners who worked under the former standard of < 10 mg dust/m(3) for 33-50 years (mean = 40 years). The distribution of retained particles within the lung compartments was markedly different between species. In all three groups of rats, 82-85% of the retained particulate material was located in the alveolar and alveolar duct lumens, primarily in macrophages. In humans, 57, 68, and 91% of the retained particulate material was located in the interstitium of the lung in the non-miners, coal miners under the current standard, and coal miners under the former standard, respectively. These results show that chronically inhaled diesel soot is retained predominantly in the airspaces of rats over a wide range of exposures, whereas in humans, chronically inhaled particulate material is retained primarily in the interstitium. In humans, the percentage of particles in the interstitium is increased with increased dose (exposure concentration, years of exposure, and/or lung burden). This difference in distribution may bring different lung cells into contact with the retained particles or particle-containing macrophages in rats and humans and may account for differences in species response to inhaled particles.

Sites of particle retention and lung tissue responses to chronically inhaled diesel exhaust and coal dust in rats and cynomolgus monkeys.

The usefulness of pulmonary carcinogenicity data from rats exposed to high concentrations of particles for quantitatively predicting lung cancer risk in humans exposed to much lower environmental or occupational concentrations has been questioned. The results of several chronic inhalation bioassays of poorly soluble, nonfibrous particles have suggested that rats may be more prone than other rodent species to develop persistent pulmonary epithelial hyperplasia, metaplasia, and tumors in response to the accumulation of inhaled particles. In addition, rats and primates differ in their pulmonary anatomy and rate of particle clearance from the lung. This paper reviews results of recent Lovelace Respiratory Research Institute (Albuquerque, NM) investigations that directly compared the anatomical patterns of particle retention and the lung tissue responses of rats and monkeys exposed chronically to high occupational concentrations of poorly soluble particles. Lung sections from male cynomolgus monkeys and F344 rats exposed 7 hr/day, 5 days/week for 24 months to filtered ambient air, diesel exhaust (2 mg soot/m3), coal dust (2 mg respirable particulate material/m3), or diesel exhaust and coal dust combined (1 mg soot and 1 mg respirable coal dust/m3) were obtained from a study conducted at the U.S. National Institute for Occupational Safety and Health and examined histopathologically and morphometrically. Within each species, the sites of particle retention and lung tissue responses were the same for diesel soot, coal dust, and combined material. Rats retained a significantly greater portion of the particulate material in the lumens of alveolar ducts and alveoli than monkeys. Conversely, monkeys retained a significantly greater portion of the particulate material in the interstitium than rats. Rats, but not monkeys, had significant alveolar epithelial hyperplastic, inflammatory, and septal fibrotic responses to the retained particles. These results suggest that anatomic patterns of particle retention and lung tissue reactions in rats may not be predictive of retention patterns and tissue responses in primates that inhale poorly soluble particles at concentrations representing high occupational exposures.

Alterations in the K-ras and p53 genes in rat lung tumors.

Activation of the K-ras protooncogene and inactivation of the p53 tumor suppressor gene are events common to many types of human cancers. Molecular epidemiology studies have associated mutational profiles in these genes with specific exposures. The purpose of this paper is to review investigations that have examined the role of the K-ras and p53 genes in lung tumors induced in the F344 rat by mutagenic and nonmutagenic exposures. Mutation profiles within the K-ras and p53 genes, if present in rat lung tumors, would help to define some of the molecular mechanisms underlying cancer induction by various environmental agents. Pulmonary adenocarcinomas or squamous cell carcinomas were induced by tetranitromethane (TNM), 4-methylnitrosamino-1-(3-pyridyl)-1-butanone (NNK), beryllium metal, plutonium-239, X-ray, diesel exhaust, or carbon black. These agents were chosen because the tumors they produced could arise via different types of DNA damage. Mutation of the K-ras gene was determined by approaches that included DNA transfection, direct sequencing, mismatch hybridization, and restriction fragment length polymorphism analysis. The frequency for mutation of the K-ras gene was exposure dependent. Only two agents, TNM and plutonium, led to mutation frequencies of > 10%. In both cases, the transition mutations formed could have been derived from deamination of cytosine. The identification of non-ras transforming genes in rat lung tumors induced by mutagenic and nonmutagenic exposures such as NNK and beryllium would help define some of the mechanisms underlying cancer induction by different types of DNA damage. Alteration in the p53 gene was assessed by immunohistochemical analysis for p53 protein and single-strand conformation polymorphism (SSCP) analysis of exons 4 to 9. None of the 93 adenocarcinomas examined was immunoreactive toward the anti-p53 antibody CM1. In contrast, 14 to 71 squamous cell carcinomas exhibited nuclear p53 immunoreactivity with no correlation to type of exposure. However, SSCP analysis only detected mutations in 2 of 14 squamous cell tumors that were immunoreactive, suggesting that protein stabilization did not stem from mutations within the p53 gene. Thus, the p53 gene does not appear to be involved in the genesis of most rat lung tumors.

Animal models of beryllium-induced lung disease.

The inhalation Toxicology Research Institute (ITRI) is conducting research to improve the understanding of chronic beryllium disease (CBD) and beryllium-induced lung cancer. Initial animal studies examined beagle dogs that inhaled BeO calcined at either 500 or 1000 degrees C. At similar lung burdens, the 500 degrees C BeO induced more severe and extensive granulomatous pneumonia, lymphocytic infiltration into the lung, and positive Be-specific lymphocyte proliferative responses in vitro than the 1000 degrees C BeO. However, the progressive nature of human CBD was not duplicated. More recently, Strains A/J and C3H/Hej mice were exposed to Be metal by inhalation. This produced a marked granulomatous pneumonia, diffuse infiltrates, and multifocal aggregates of interstitial lymphocytes with a pronounced T helper component and pulmonary in situ lymphocyte proliferation. With respect to lung cancer, at a mean lung burden as low as 17 micrograms Be/g lung, inhaled Be metal induced benign and/or malignant lung tumors in over 50% of male and female F344 rats surviving > or = 1 year on study. Substantial tumor multiplicity was found, but K-ras and p53 gene mutations were virtually absent. In mice, however, a lung burden of approximately 60 micrograms (-300 micrograms Be/g lung) caused only a slight increase in crude lung tumor incidence and multiplicity over controls in strain A/J mice and no elevated incidence in strain C3H mice. Taken together, this research program constitutes a coordinated effort to understand beryllium-induced lung disease in experimental animal models.

Ultraviolet radiation, solar dermatosis, and cutaneous neoplasia in beagle dogs.

Beagle dogs that were part of a life span study of the effects of low-level ionizing radiation during development were evaluated for the incidence of skin neoplasia and solar dermatosis. A total of 991 dogs up to 14 years of age were examined. The dogs were housed in gravel-based, outdoor pens with doghouses in a high-altitude, high-sunshine level environment. Solar dermatosis was restricted to the sparsely haired, nonpigmented abdominal skin. Skin neoplasms were either removed surgically or found at necropsy. Solar dermatosis was diagnosed in 363 of the 991 dogs, an incidence of 36.6%. There were 175 hemangiomas, hemangiosarcomas, or squamous cell carcinomas of the skin in the 991 dogs. Of these, 129 tumors occurred in dogs with, and only 46 in dogs without, solar dermatosis. Of the dogs with solar dermatosis, 93 (26%) had at least one of the three tumor types, compared to only 44 (7%) of dogs without solar dermatosis. Thirty-two dogs had multiple tumor types and solar dermatosis, compared to only two dogs with multiple tumor types and no solar dermatosis. There was a highly significant correlation (P less than 0.001) between the occurrence of these tumor types and solar dermatosis in the unpigmented abdominal skin. This correlation was strongest for the malignant neoplasms. Whole-body gamma-radiation exposures were delivered at one of three prenatal or three postnatal ages up to 1 year of age. There appeared to be an increased risk for hemangiosarcomas and squamous cell carcinomas in dogs with solar dermatosis and given gamma-ray exposures at 1 year of age. This suggests an interaction between exposures to ionizing and ultraviolet radiation.

Analysis of the K-ras and p53 pathways in X-ray-induced lung tumors in the rat.

The risk from exposure to low-dose radiation in conjunction with cigarette smoking has not been estimated due in part to limited knowledge surrounding the molecular mechanisms underlying radiation-induced cancers. The purpose of this investigation was to determine the frequency for alterations in genes within the K-ras and p53 signal and cell cycle regulatory pathways, respectively, in X-ray-induced lung tumors in the F344/N rat. These tumors were examined for genetic alterations in the K-ras, c-raf-1, p53, mdm2 and cip1 genes. No K-ras mutations were detected by sequencing in 18 squamous cell carcinomas (SCCs) or 17 adenocarcinomas. However, using a K-ras codon 12 mutation selection assay, a codon 12 GGT --> GAT mutation was detected in one SCC, suggesting that activation of the K-ras proto-oncogene is both a rare and late event. Single-strand conformation polymorphism (SSCP) analysis of the kinase-binding domain of the c-raf-1 gene did not detect any polymorphisms. Three of 18 SCCs but none of the adenocarcinomas showed p53 nuclear immunoreactivity. Single-strand conformation polymorphism analysis of exons 4-9 of the p53 gene detected only an exon 9 mutation in one SCC. Mutations were not detected in the three SCCs with immunoreactive p53 protein. No amplification of the mdm2 gene was detected; however, nuclear mdm2 immunoreactivity was present in one of the three SCCs that stained positive for the p53 protein. Thus the increased level of p53 protein in one SCC may stem from stabilization by the mdm2 gene product. The complete cDNA of the rat cip1 gene comprising 810 bases was cloned and sequenced. Overall homology between the rat and human cip1 genes was 74%. Homology between the rat and mouse genes was 90%. The frequency of somatic mutations in exon 2 of the cip1 gene was determined by SSCP analysis. No alterations in electrophoretic mobility were detected. The results of this investigation indicate that alterations in the K-ras and p53 pathways do not play a major role in the genesis of X-ray-induced lung tumors in the rat.

Biological effects of 137CsCl injected in beagle dogs.

The toxicity of intravenously administered 137CsCl in the beagle dog was investigated as part of a program to evaluate the biological effects of internally deposited fission-product radionuclides. The intravenous route of exposure was chosen for simplicity and accuracy because it was known that after intravenous injection, inhalation or ingestion, internally deposited 137CsCl is rapidly absorbed and distributed throughout the body, exposing the whole body to beta-particle and gamma radiations. Fifty-four dogs were injected intravenously with 137Cs to provide one group of six dogs with mean initial body burdens of 141 MBq 137Cs/kg body mass and four groups of 12 dogs each with mean initial body burdens of 104, 72, 52 and 36 MBq 137Cs/kg. Twelve dogs were injected with isotonic saline as study controls. Because the number of study control dogs was small, data from an additional 49 control dogs from other studies at the Inhalation Toxicology Research Institute that were performed over a similar span of years were also used. There was a significant, dose-dependent decrease in survival of the 137Cs-injected dogs. Eleven 137Cs-injected dogs, including all six in the highest initial body burden group, died within 81 days after injection, primarily due to hematopoietic cell damage resulting in severe pancytopenia. An additional 25 dogs had transient hematological dyscrasia but survived for long times. All 137Cs-injected male dogs had marked damage to the germinal epithelium of the testicular seminiferous tubules with azoospermia in the long-term survivors. Benign and malignant neoplasms occurred in a variety of organs in 137Cs-injected dogs, rather than in a single target organ. When individual organs were considered, the incidence of malignant neoplasms was increased in the liver and in the nasal cavity and paranasal sinuses of the 137Cs-injected dogs. There was a 137Cs treatment effect in the incidence of malignant neoplasms (P < 0.001) in male dogs but no 137Cs-related treatment effect in female dogs. However, when malignant mammary neoplasms were excluded from the analysis, there was no gender difference, and there was a dose-related response (P < 0.001) in both males and females for the incidence of malignant neoplasms.

Pulmonary carcinogenicity of relatively low doses of beta-particle radiation from inhaled 144CeO2 in rats.

This study was conducted to examine the carcinogenic effects of inhaled beta-particle-emitting radionuclides, particularly in lower dose regions in which there were substantial uncertainties associated with available information. A total of 2751 F344/N rats (1358 males and 1393 females) approximately 12 weeks of age at exposure were used. Of these, 1059 rats were exposed to aerosols of 144CeO2 to achieve mean desired initial lung burdens (ILBs) of 18 kBq (low level), 247 rats to achieve mean ILBs of 60 kBq (medium level) and 381 rats to achieve mean ILBs of 180 kBq (high level). Control rats (total of 1064) were exposed to aerosols of stable CeO2. Based on the 95% confidence intervals of the median survival times and the cumulative survival curves, there were no significant differences in the survival of groups of female and male exposed rats relative to controls. The mean lifetime beta-particle doses to the lungs of the rats in the four groups were: low level, 3.6 +/- 1.3 (+/-SD) Gy; medium level, 12 +/- 4.5 Gy; and high level, 37 +/- 5.9 Gy. The crude incidence of lung neoplasms increased linearly with increasing doses to the lungs (controls, 0.57%; low level, 2.0%; medium level, 6.1%; and high level, 19%). The estimated linear risk coefficients for lung neoplasms per unit of dose to the lung were not significantly different for the three dose levels studied. The risk coefficient at the lower level was 39 +/- 14 (+/-SE) excess lung neoplasms per 10(4) rat Gy; at the medium level the risk was 47 +/- 12; and at the higher level the risk was 50 +/- 9.0. The relationship of beta-particle dose to the lung and the crude incidence of lung neoplasms was described adequately by a linear function. We concluded that the risk of lung neoplasms in rats per unit of radiation dose did not increase with decreasing mean beta-particle dose to the lung over the range of 3.6 to 37 Gy. The weighted average of these three values was 47 +/- 6.4 (+/-SE) excess lung neoplasms per 10(4) rat Gy. To extend the risk coefficients for lung neoplasms to lower doses by experimentation will require much larger numbers of rats than used in this study.

Biological effects of 137CsCl injected in beagle dogs of different ages.

The toxicity of 137Cs in the beagle dog was investigated at the Inhalation Toxicology Research Institute (ITRI) and Argonne National Laboratory (ANL) as part of programs to evaluate the biological effects of both radionuclides in atomic bomb fallout and internally deposited fission-product radionuclides. In the ITRI study, young adult dogs were exposed once by intravenous injection to a range of 137Cs concentrations; the results have recently been published (Nikula et al., Radiat. Res. 142, 347-361, 1995). The purpose of the present report is to summarize the ANL study and to compare the results of the two studies. At ANL, 63 dogs in three age groups (15 juveniles, 142-151 days old; 38 young adults, 388-427 days old; and 10 middle-aged dogs, 1387-2060 days old) were given 137Cs intravenously at levels (61-162 MBq/kg) near those expected to be lethal within 30 days after injection. There were 17 control dogs from the same colony. Twenty-three of the dogs injected with 137Cs, including all middle-aged dogs, died within 52 days after injection due to hematopoietic cell damage resulting in severe pancytopenia that led to fatal hemorrhage and/or septicemia. The other significant early effect was damage to the germinal epithelium of the seminiferous tubules of all male dogs. These early effects are the same as those reported for the dogs injected with 137Cs at ITRI. In addition, the design of the ANL study revealed an age- and gender-related differential radiosensitivity for early effects: The middle-aged dogs died significantly earlier due to complications of hematological dyscrasia compared to the juvenile and young adult dogs, and the middle-aged females died significantly earlier than the middle-aged males. The most significant non-neoplastic late effects in the 137Cs-injected dogs from ANL and ITRI were atrophy of the germinal epithelium of seminiferous tubules with azoospermia, and a significant dose-dependent decrease in survival. However, the survival of the ANL dogs was decreased more than that of the ITRI dogs at similar radiation doses from 137Cs. Numerous neoplasms occurred at many different sites in the dogs injected with 137Cs at ANL and ITRI. Two differences in the findings of the two studies were that (1) there was an increased risk for malignant thyroid neoplasms in the ANL male dogs injected with 137Cs, but not the ITRI dogs of either gender, and (2) there was an increased relative risk for benign neoplasms excluding mammary neoplasms in the ITRI dogs injected with 137Cs, but not the ANL dogs. In both groups, there were dose-related increased incidences of malignant neoplasms, malignant neoplasms excluding mammary neoplasms, all sarcomas considered as a group, all non-mammary carcinomas considered as a group and malignant liver neoplasms. In summary, the similarity of the findings between the two studies and the dose-response relationships for survival and for large groupings of neoplasms suggests that these results are consistent findings in 137Cs-injected dogs and might be dose-related late effects in humans exposed to sufficient amounts of internally deposited 137Cs.

Dose-response relationships between inhaled beryllium metal and lung toxicity in C3H mice.

Inhaled beryllium (Be) can induce a range of adverse pulmonary responses in animals and humans including acute pneumonitis, chronic granulomatous lung disease, and cancer. To facilitate comparisons with our previous data describing Be toxicity in rats, we evaluated the toxic effects of inhaled Be metal in mice. Groups of 34 strain C3H/HeJ mice were acutely exposed by the nose-only route to aerosolized Be metal to achieve measured initial lung burdens of 0, 1.7, 2.6, 12, or 34 microg. All mice received aerosolized 85 Sr-labeled fused aluminosilicate particles (85 Sr-FAPs) immediately before their Be exposure so that the influence of Be on lung retention of these poorly soluble tracer particles could be externally quantitated. Groups of mice were euthanized at 8, 15, 40, 90, 210, and 350 days after exposure for evaluation of histopathological changes and for cytologic and biochemical indicators of lung damage measured in bronchoalveolar lavage fluid. Clearance of 85 Sr-FAP tracer particles through 196 days after exposure was delayed in mice receiving the 12 and 34 microg Be lung burdens, but not the 1.7 or 2.6 microg lung burdens. Increased total cell numbers, increased percentage of neutrophils, and elevated levels of total protein and the activities of beta-glucuronidase and lactate dehydrogenase in bronchoalveolar lavage fluid were observed in the two highest Be lung burden groups compared with controls. Lung lesions included particle-containing macrophages, granulomatous pneumonia, lymphocytic interstitial aggregates, and mononuclear interstitial infiltrates. These lesions were occasionally seen in mice receiving the 2.6 microg lung burden, were present in most of the mice receiving 12 or 34 microg lung burdens, and were generally increased in severity with time and lung burden. Thus, we have demonstrated that a single, acute inhalation exposure to Be metal can chronically retard particle clearance and induce lung damage in mice. The initial lung burdens used caused responses ranging from no apparent effects to significant Be-induced responses. A comparison of these data with our previous data from rats indicates that the mass of Be metal required to induce lung damage in mice is similar to that needed for rats. When expressed on a lung weight-normalized basis, mice appeared to be more resistant to the toxic effects of inhaled Be than rats.

Cigarette smoke exposure produces more evidence of emphysema in B6C3F1 mice than in F344 rats.

Cigarette smoke (CS) causes pulmonary emphysema in humans, but results of previous studies on CS-exposed laboratory animals have been equivocal and have not clearly demonstrated progression of the disease. In this study, morphometry and histopathology were used to assess emphysema in the lungs of B6C3F1 mice and Fischer-344 rats. The animals were exposed, whole-body, to CS at a concentration of 250 mg total particulate matter/m3 for 6 h/day, 5 days/week, for either 7 or 13 months. Morphometry included measurements of parenchymal air space enlargement (alveolar septa mean linear intercept [Lm], volume density of alveolar air space [VVair]), and tissue loss (volume density of alveolar septa [VVspt]). In addition, centriacinar intra-alveolar inflammatory cells were counted to assess species differences in the type of inflammatory response associated with CS exposure. In mice, many of the morphometric parameters indicating emphysema differed significantly between CS-exposed and control animals. In CS-exposed rats, only some of the parameters differed significantly from control values. The Lm in both CS-exposed mice and rats was increased at 7 and 13 months, indicating an enlargement of parenchymal air spaces, but the VVair was increased significantly only in CS-exposed mice. The VVspt was decreased at both time points in mice, but not in rats, indicating damage to the structural integrity of parenchyma. Morphologic evidence of tissue destruction in the mice included alveoli that were irregular in size and shape and alveoli with multiple foci of septal discontinuities and isolated septal fragments. Morphometric differences in the mice at 13 months were greater than at 7 months, suggesting a progression of the disease. Inflammatory lesions within the lungs of mice contained significantly more neutrophils than those lesions in rats. These results suggest that B6C3F1 mice are more susceptible than F344-rats to the induction of emphysema by this CS exposure regimen and that in mice the emphysema may be progressive. Furthermore, the type of inflammatory response may be a determining factor for species differences in susceptibility to emphysema induction by CS exposure.

Pharmacokinetics of ICG and HPPH-car for the detection of normal and tumor tissue using fluorescence, near-infrared reflectance imaging: a case study.

We present in vivo fluorescent, near-infrared (NIR), reflectance images of indocyanine green (ICG) and carotene-conjugated 2-devinyl-2-(1-hexyloxyethyl) pyropheophorbide (HPPH-car) to discriminate spontaneous canine adenocarcinoma from normal mammary tissue. Following intravenous administration of 1.0 mg kg-1 ICG or 0.3 mg kg-1 HPPH-car into the canine, a 25 mW, 778 nm or 70 mW, 660 nm laser diode beam, expanded by a diverging lens to approximately 4 cm in diameter, illuminated the surface of the mammary tissue. Successfully propagating to the tissue surface, ICG or HPPH-car fluorescence generated from within the tissue was collected by an image-intensified, charge-coupled device camera fitted with an 830 or 710 nm bandpass interference filter. Upon collecting time-dependent fluorescence images at the tissue surface overlying both normal and diseased tissue volumes, and fitting these images to a pharmacokinetic model describing the uptake (wash-in) and release (wash-out) of fluorescent dye, the pharmacokinetics of fluorescent dye was spatially determined. Mapping the fluorescence intensity owing to ICG indicates that the dye acts as a blood pool or blood persistent agent, for the model parameters show no difference in the ICG uptake rates between normal and diseased tissue regions. The wash-out of ICG was delayed for up to 72 h after intravenous injection in tissue volumes associated with disease, because ICG fluorescence was still detected in the diseased tissue 72 h after injection. In contrast, HPPH-car pharmacokinetics illustrated active uptake into diseased tissues, perhaps owing to the overexpression of LDL receptors associated with the malignant cells. HPPH-car fluorescence was not discernable after 24 h. This work illustrates the ability to monitor the pharmacokinetic delivery of NIR fluorescent dyes within tissue volumes as great as 0.5-1 cm from the tissue surface in order to differentiate normal from diseased tissue volumes on the basis of parameters obtained from the pharmacokinetic models.

In vitro exposure of tracheobronchial epithelial cells and of tracheal explants to ozone.

An in vitro system for exposing respiratory epithelial cells or explant tissues to ozone has been developed and characterized. This system is designed to generate and monitor consistent, reproducible levels of ozone, over a range of concentrations, in a humidified atmosphere, and to allow an exposure time of 24 h or longer. Based on chemical analysis, highly reproducible concentrations of ozone are delivered throughout the chamber, with a coefficient of variation of < 5% between five replicate vials exposed to 0.5 ppm of ozone for 50 min. The viability of cultured human tracheobronchial epithelial cells, as measured by the ability to oxidize a vital dye, and of rat tracheal epithelium, as measured by total numbers of necrotic cells in tracheal explants, after ozone exposure was examined in this system. Responses of cultured cells to ozone exposure as measured by bioassay were consistent with the observed low level of variability of ozone concentration between replicate incubation dishes or vials. Responses of cultured cells to ozone were proportional to duration of exposure and inversely proportional to the volume of medium covering the cells. We conclude that this newly developed in vitro exposure system will allow relatively simple and convenient exposure of cultured cells or organs to ozone or other gaseous agents under highly controlled and reproducible conditions.

A microassay for measuring cytosine DNA methyltransferase activity during tumor progression.

The cytosine DNA methyltransferase (MT) enzyme, which catalyzes DNA methylation at CpG sites, is overexpressed at the mRNA level during the progressive stages of colon cancer. This paper describes the adaption of a sensitive microassay for determining MT enzyme activity during tumor progression in human colon and murine lung. MT activity was progressively elevated in mucosa from familial adenomatosis polyposis patients, mucosa adjacent to cancers, and in colonic adenocarcinomas when compared to colonic mucosa from control patients. In addition, the activity of this enzyme was increased in alveolar type II but not Clara cells isolated from A/J mice following carcinogen exposure and continued to increase during tumor progression. The use of a microassay for measuring MT activity indicates that changes in enzyme activity were in general agreement with previous findings of increased MT mRNA levels during colon cancer progression and also implicates the involvement of this pathway in lung cancer development.

Pulmonary delivery of nanoparticles of insoluble, iodinated CT X-ray contrast agents to lung draining lymph nodes in dogs.

Lung cancer continues to be a leading cause of death around the world. Staging of this disease is critically dependent upon the involvement or noninvolvement of the lymph nodes which drain the region of lung containing the lesion/tumor. Palpation, unenhanced CT, and lymph node excision (i.e., mediastinectomy) are currently used to ascertain the status of these regional draining lymph nodes. The work reported herein details the first efforts toward the pulmonary instillation of iodinated nanoparticles for contrast-enhanced CT of lung draining lymph nodes. The data reflect the impact of dose, time post instillation, and formulation (surfactant) upon the observed CT enhancement of the tracheobronchial lymph nodes of beagle dogs. In addition, initial safety is discussed with both macroscopic and microscopic observations. The results indicate that pulmonary instillation of small volumes of iodinated nanoparticles could be successfully used to aid staging of lung cancer by CT imaging.

CT imaging of intrathoracic lymph nodes in dogs with bronchoscopically administered iodinated nanoparticles.

RATIONALE AND OBJECTIVES: The purpose of the study was to determine if airway instillation of iodinated nanoparticles results in contrast material enhancement of tracheobronchial lymph nodes in dogs. MATERIALS AND METHODS: Eight dogs underwent intrabronchial instillation of iodinated nanoparticles; six dogs received 900 mg each, and two dogs received 450 mg each. Spiral computed tomography (CT) was then performed 2-34 days later. RESULTS: CT scans obtained 2 days after instillation showed the presence of contrast material within the lung parenchyma but no nodal enhancement. Scans obtained 6-34 days after instillation showed enhancement of the right, left, and middle tracheobronchial lymph nodes (analogous to the mediastinal nodes in humans). Mean nodal attenuation on CT images was 117 HU +/- 43, and the mean nodal volume was 129 mm3 +/- 113. Histologic specimens of the nodes showed macrophage hyperplasia. CONCLUSION: Iodinated nanoparticles instilled into small airways are transported to the tracheobronchial lymph nodes, where they result in contrast enhancement.